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WO2003035895A2 - Analyse a parametres multiples d'acides nucleiques complets et de caracteristiques morphologiques sur le meme echantillon - Google Patents

Analyse a parametres multiples d'acides nucleiques complets et de caracteristiques morphologiques sur le meme echantillon Download PDF

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WO2003035895A2
WO2003035895A2 PCT/US2002/034570 US0234570W WO03035895A2 WO 2003035895 A2 WO2003035895 A2 WO 2003035895A2 US 0234570 W US0234570 W US 0234570W WO 03035895 A2 WO03035895 A2 WO 03035895A2
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cell
rna
group
cells
analysis
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PCT/US2002/034570
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WO2003035895A3 (fr
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Shawn Mark O'hara
Daniel Zweitzig
Brad Foulk
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Immunivest Corporation
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Priority to JP2003538395A priority Critical patent/JP2005507997A/ja
Priority to IL16135702A priority patent/IL161357A0/xx
Priority to KR10-2004-7006181A priority patent/KR20040047971A/ko
Priority to EP02795565A priority patent/EP1438419A2/fr
Priority to BRPI0213520-5A priority patent/BR0213520A/pt
Priority to CA002465288A priority patent/CA2465288A1/fr
Publication of WO2003035895A2 publication Critical patent/WO2003035895A2/fr
Publication of WO2003035895A3 publication Critical patent/WO2003035895A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

  • This invention relates generally to gene specific amplification, analysis and profiling of cytosolic biomolecules useful in the fields of oncology and diagnostic testing.
  • the invention is particularly useful in such fields as cancer screening, selecting and monitoring for chemotherapy treatment, or cancer recurrence. More specifically, the present invention provides methods, apparatus, and kits to facilitate comprehensive analysis of mRNA and DNA from tumor cells, or other rare cells from biological samples while simultaneously maintaining cell integrity for enumeration and morphological image analysis.
  • the invention also provides methods that permit the analysis of soluble cytosolic biomolecules releasable from a cell, such as a tumor cell, by means of permeabilizing reagents for determining expression profiles of the released nucleic acids, while still maintaining the morphological and antigenic characteristics of cells for subsequent or parallel muitiparameter flowcytometric, image, and immunocytochemical analyses (see US 6,365,362).
  • the invention also provides methods that enable the same comprehensive analyses using stabilized samples from aldehyde and aldehyde-urea derivative based fixatives.
  • Any given cell will express only a fraction of the total number of genes present in its genome. A portion of the total number of . genes that are expressed determine aspects of cell function such as development and differentiation, homeostasis, cell cycle regulation, aging, apoptosis, etc. Alterations in gene expression decide the course of normal cell development and the appearance of disease states, such as cancer. The expression of specific genes will have a profound effect on the nature of any given cell. Accordingly, the methods of analyzing gene expression, as such as those provided by the present invention, are important in basic molecular biological research and in tumor biology.
  • Differential gene expression is a commonly used method of assessing gene expression in a cell.
  • cDNA microarray analysis compares cDNA target sequence levels obtained from cells or organs from healthy and diseased individuals. These targets are then hybridized to a set of probe fragments immobilized on a membrane. Differences in the resultant hybridization pattern are then detected and related to differences in gene expression of the two sources (US 6,383,749). This procedure requires slow and time-consuming analysis of several hundred thousand gene-specific probes.
  • competing events such as interactions between non- complementary target sequences nonspecific binding between target and probe and secondary structures in target sequences may interfere with hybridization resulting in a decline in the signal-to-noise.
  • Gene specific primer sets have been described in assaying differential expression (US 5,994,076 and US 6,352,829). Here, gene specific primer sets were used to specifically amplify mRNA library subsets in complex libraries achieving a cDNA array signal' improvement when compared to whole library labeling amplification. The focus of this type of analysis was to compare sample array expression profiles as part of gene discovery research, not development of methods for practical cellular RNA analysis with utility in diagnostics.
  • DLBCL Diffuse Large B Cell Lymphoma
  • the present invention provided methods, apparatus, and kits for assessing gene expression in amplified mRNA isolated from circulating rare cells (see Figure 1 ) which overcome the disadvantages of the prior art techniques which are described above.
  • the present invention provides methods of isolating soluble or releasable cytoplasmic biomolecules from a single target cell or a cell population while maintaining structural cell integrity or phenotypic characteristics. Accordingly, cell(s) are either fresh or stabilized and fixed with a cross-linking agent, contacted with a pore-forming permeabilization composition, and the nucleic acids recovered.
  • RNA from stabilized cells is recovered via combinations of proteinase and nucleophiles reversal agents either for amplification and subsequent qualitative and quantitative PCR analysis or for quantitative analysis via gene specific subsets of reverse transcription (RT) primers fused with and followed by universal primer PCR amplification.
  • RT reverse transcription
  • the present invention is also directed to separating nuclear and/or mitochondrial DNA, RNA, proteins and other soluble components within a targeted cell by contacting a cell or cell population with a permeabilization composition and separately analyzing the released and/or unreleased fraction for one or more constituents such as the nuclear and/or mitochondrial DNA, total RNA, mRNA, soluble proteins, and other target substances (US App. 60/330,669).
  • the present invention incorporates the analysis of both cytoplasmic biomolecules and membrane or surface biomolecules from the same cell(s) or cell population by contacting the cell(s) with a permeabilization composition and separately analyzing the cytoplasmic biomolecules and the surface biomolecules to generate functional cell profiles encompassing characteristics derived from genotypic and phenotypic cell characteristics for differentiating normal from transformed cells.
  • CTC circulating tumor cells
  • T7RNAP T7 RNA polymerase
  • Pre-amplification is completed by linear amplification of the entire mRNA library using modifications of the Eberwine aRNA method (Van Gelder et al. 1990).
  • generation of an anti-sense mRNA library (aRNA) library preamplificaiton results in at least a thousandfold increase of all messages present in the original mRNA isolated from ferrofluid enriched circulating cells.
  • Gene specific primers are then used to amplify only the gene panel of interest. These primers are designed to amplify transcripts indicative of known rare events like circulating tumor cells.
  • the number of target sequences can be as small as two or as large as necessary to allow correlation with some indicative characteristic of the rare event. This can occur as separate individual reactions or within a single reaction vial.
  • Subsequent analysis yields at least a qualitative assessment of the target sequences and is achieved with methods such as, but not limited to, one of two types of multigene analysis methods we present here as gene specific primed (GSP) arrays and/or GSP sets-RT (universal PCR).
  • GSP gene specific primed
  • Universal PCR achieves multigene analysis from sample recovered mRNA in a single reaction tube with or without mRNA library preamplification. No preamplification allows only one panel of genes to be analyzed at one time. Preamplification adds the advantage of analyzing a single sample in up to 1000 different reactions, thus many different panels of genes can be interrogated at different times. While it will be noted that other methods are available, analysis of universal PCR cocktail panels is accomplished by array or capillary gel electrophoresis (CGE). The system allows, therefore, for both a quantitative and qualitative determination of 1 to thousands of separate mRNA types simultaneously when measured in cDNA microarray format.
  • CGE capillary gel electrophoresis
  • the present invention includes a combination of the above mentioned isolation and profiling analysis directed to protocols and kits comprising some or all necessary reagents including a permeabilization composition, RNA recovery after cross-linking, magnetic microspheres with oligo(dT) probes covalently bound to the surface, and other gene specific magnetic microsphere-bound probes for capture and analysis of comprehensive RNA analysis using a small or large microarray, capillary gel electrophoresis (CGE), HPLC, electrophoresis and other analytical platforms.
  • CGE capillary gel electrophoresis
  • Figure 1 shows a flowchart depicting the variety of capabilities and options enabled by the inventions described in this application for muitiparameter analysis on a single sample. Phenotypic and genotypic analysis is obtained on fixed or non-fixed cells.
  • Figure 2 shows the reverse image (negative) of denatured total RNA analyzed by 2% agarose gel electrophoresis after SYBR Gold staining of about 1600 SKBR3 breast cancer cells that were Immuniperm-treated for various times before total RNA was isolated from the resultant supernatant via Trizol plus pellet paint co-precipitate.
  • Figure 3 shows a 1% denaturing total RNA agarose gel stained with ethidium bromide comprising whole cells, Immuniperm (saponin)-permeabilized cells from the cell pellet fraction, and cells from the supernatant fraction of ImmunipermCB permeabilized SKBR3 breast cancer cells.
  • Figure 4 shows a phosphor image of a Northern blot of the gel shown in Figure 3 hybridized by a polynucleotide kinase treated 32 P-labeled oligo(dT) (25 mer) probe.
  • the radioactive signals correspond to all the poly(A)+ mRNA transcripts of the total RNA which was derived from whole cells and the two Immuniperm® treated cell fractions from the gel shown in Figure 3.
  • Figure 5 shows a Northern blot from Figure 4 stripped through conventional dissociation and removal of the labeled oligo(dT) probe and re-probed with a nuclear-specific precursor rRNA probe.
  • Figure 6 shows the Northern blot, from Figure 5, that was stripped and re- probed with mitochondrial-specific 12s rRNA probe.
  • Figure 7 shows a cDNA array dot blot hybridization pattern comparison when the corresponding mRNA, used to generate the gel images in Figure 3, is alpha- 32 P-nucleotide labeled during first strand oligo(dT) primed cDNA synthesis. Labeled first strand cDNA was then used as the hybridization probe. Pattern comparison shows the same relative abundance of mRNA exists in all three RNA cell fractions.
  • Figure 8 shows the gel image of the relative cytosolic total RNA, both quantitatively and qualitatively, obtained after separately treating multiple aliquots containing about 770 PC-3 cells each with Immuniperm®.
  • Figure 9 shows the preservation, recovery and RNA integrity analysis of 90- 100% of the total RNA library using CytoChexTM and other aldehyde based fixatives followed by enzyme digestion.
  • mass normalized portions of 300,000 SKBR3 cell line cells which were first spiked into freshly drawn 7.5ml peripheral blood (EDTA Vacutainer tube) in both control lanes without (phosphate buffered saline, PBS) and with three different fixatives being Cyto-ChexTM, StabilcyteTM and TransfixTM. After mixing these were allowed to incubate at room temperature (20-25°C) for 24 hours. After which the SKBR3 cells were enriched from the blood using VU-1 D9 (EpCAM)- ferrofluid immunomagnetic selection.
  • VU-1 D9 EpCAM
  • RNA isolations were separated with a 1% denaturing agarose, stained with SYBR Gold, alpha imager densitometry imaged and then Northern Blotted and finally oligo(dT) probed to show relative quality and quantity of respective total RNA and mRNA libraries recovered.
  • Figure 10A & 10B shows relative rate of Cyto-ChexTM, StabilcyteTM, TransfixTM, paraformaldehyde, formaldehyde, glutaraldehyde and glyoxal fixation over a 1 , 2, and 4 hour time course.
  • the relative- rate time course of Cyto-ChexTM, StabilcyteTM, TransfixTM, paraformaldehyde, formaldehyde, glutaraldehyde and glyoxal fixation were evaluated at 1 , 2, and 4 hours. Samples of 7.5 ml of blood were prepared form a single donor by the same method as described in Figure 9.
  • RNA isolations were selected and processed for RNA isolation at 1 , 2, and 4 hour time end points.
  • the resultant normalized RNA isolations were separated with a 1% denaturing agarose, stained with SYBR Gold, alpha imager densitometry imaged and then Northern Blotted and finally oligo(dT) probed to show relative quality and quantity of respective total RNA and mRNA libraries recovered.
  • Figure 10C shows relative rate of fixation of Cyto-ChexTM vs. paraformaldehyde over a 15, 30, and 45 min time course.
  • the relative-rate time course of fixation Cyto-Chex vs. paraformaldehyde were evaluated at 15, 30, and 45 min.
  • Samples of 7.5 ml of blood were prepared from a single donor by the same method as described in Figure 9. The only difference is that they were selected at 15, 30, and 45 min time end points.
  • Figure 11 shows the effects of variations on nucleophile and enzyme on the quality and quantity of RNA recovery from Cyto-ChexTM preservation supporting that when used in combined treatments for improved sequence analysis quality is likely.
  • the resultant normalized RNA isolations were separated with a 1% denaturing agarose, stained with SYBR Gold, alpha imager densitometry imaged and then Northern Blotted and finally dT probed to show relative quality and quantity of respective total RNA and mRNA libraries recovered.
  • Figure 12A shows the feasibility of diagnostic applications demonstrated by detection of specific mRNA From 10 SKBR3 cells/7.5ml Blood in 24hr Cyto- ChexTM stabilized blood with proteinase recovery and aRNA preamplification. Feasibility of diagnostic applications are here demonstrated by sensitive and reproducible detection of specific mRNA from triplicate 10 or 20 SKBR3 cells spiked into 7.5ml peripheral blood. The spiked blood was stabilized immediately treated with Cyto-Chex to stabilize the cellular RNA. After incubating for one day at room temperature (20-25C) the stabilized cells were selectively enriched using VU-1 D9 (EpCam)-Ferro Fluid Immunomagnetic selection. Enrichment was followed by proteinase K digestion to liberate the RNA so that silica binding RNA isolation followed by aRNA preamplification and gene specific quantitative RT-PCR could be performed for CK19 and EpCAM.
  • VU-1 D9 EpCam
  • Figure 12B and 12C shows CK19 and EpCAM respective Q-PCR from SKBR Cell Spike, ferrofluid selection, and CytoChexTM treatment, followed by proteinase reversal.
  • This experiment shows the results of the quantitative RT- PCR analysis, which was normalized to original total RNA mass prior to graphing. Thus, the values shown are equivalent to the original mRNA population contained in the original RNA isolation prior to aRNA amplification.
  • Figure 12D and 12E shows CD19 and EpCAM respective Q-PCR on aRNA derived from SKBR cells treated with cell stability reagents. These experiments show the RNA derived from the three different fixatives, which were shown in Figure 9 Cyto-ChexTM, StabilcyteTM and TransfixTM.
  • Figure 13A shows RT-PCR amplification efficiency of a CK19-cRNA standard containing the 3'-most 800 base sequence of the CK19 mRNA transcript.
  • Serial two-fold dilutions of the CK19-cRNA standard containing 200, 100, 50, 25, 12,5 copies were spiked into 2 ng of CK19 negative total RNA from white blood cells in triplicate resulting in a maximum coefficient of variation of 27%. Standard deviation bars are shown. Dilutions of cRNA to less than one copy and no template controls did not produce detectable signals.
  • Figure 13B shows the relative RT-PCR gene expression levels after agarose gel electrophoresis.
  • CK19 cRNA was spiked into total RNA from white blood cells at levels of 25 copies, 250 copies, 2,500 copies and 25,000 copies in panel 1 , panel 2, panel 3, and panel 4, respectively.
  • Figure 13C compares the relative representation in the same mRNA library of unamplified and T7 promoter-based amplified mRNA transcripts. Relative abundance was assessed by examining 8 different mRNA transcripts (PSA, PSM, MGB1 , MGB2, CK8, CK19, PIP, EpCam) using the RT-PCR kinetic curve method.
  • Figure 14A shows scatter plot bar graphs of a survey of genes indicating the presence of circulating epithelial cells. Human blood, immunomagnetically enriched for cells expressing the EpCAM antigen on their cell surface, the samples were first aRNA preamplified and then 25ng were reverse transcribed.
  • Figure 14B depicts a survey of genes indicating prostate tumor organ of origin status via the same methods as described in 14A.
  • Figure 14C depicts a survey of genes indicating the presence of therapeutic target status via the same methods as described in 14A.
  • Figure 15A, 15B, and 15C show individual HRPC patient longitudinal monitoring of CTC and RT-PCR multigene analysis before, during, and after new line chemotherapy.
  • the x-axis shows sampling time in weeks
  • the left y- axis shows the CTC level with the solid circle symbol.
  • the right y-axis shows the relative mRNA expression levels with corresponding symbols of open- square for Androgen Receptor (AR), open circle for Hepsin (HPN) and open triangle for multidrug resistance (MDR1).
  • AR Androgen Receptor
  • HPN open circle for Hepsin
  • MDR1 multidrug resistance
  • Relative mRNA levels are illustrated here during treatment courses of Lupron alone as shown Figure 15A, and 2 patients being treated with Lupron combined with administration of doses of Taxotere and Estramustine chemotherapy symbolized by the vertical arrows on the x-axis (Tx/Ex) in Figure 15B and Figure 15C. Bars on top indicate long term hormonal ablation treatment was on going.
  • pre-analytical cell preparation could also be limited to soluble cytoplasmic RNA, total cellular RNA, total cellular DNA, and/or proteins, having the major objective to homogenize cells in order to release soluble intracellular components (US 6,329,179).
  • traditional phenotypic characterizations required fixation of cell structures achieved through exposure of cells to a cross-linking agent, such as paraformaldehyde, formaldehyde, glutaraldehyde, etc. These harsh cell fixation conditions simultaneously cause undesirable covalent crosslinking and/or fragmentation of all the isolatable RNA species.
  • RNAIaterTM (Ambion) is commercially available RNA stabilization solution, which stabilizes RNA but does not allow immunomagnetic, immunochemistry or image analysis on the same sample and is not effective for blood.
  • PreAnalytiX offers a blood RNA stabilizer but is nothing more than the chaotropic agent guanidine isothiocyanate solution (GITC) solution in a VacutainerTM tube enabling nothing more than traditional homogenization based solely on total RNA isolation.
  • GITC guanidine isothiocyanate solution
  • mRNA recovered from fixed cells is not quantitative and is severely degraded or fragmented reducing the size of intact RNA with an average size of approximately 1750 bases as much as ten-fold to a highly variable average size of approximately 200 bases, and contains many complex chemical modifications, which are not well understood.
  • fixative derived RNA is severely compromised mRNA analysis (Current Protocols in Molecular Biology, Wiley, (2002)).
  • Tedious non- quantitative mRNA salvage techniques combined with reverse transcriptase polymerase chain reaction (RT-PCR) analysis designed for amplicons of less than 100 base pairs in length show limited value, albeit in a qualitative not quantitative manner (US 5,346,994). Further, this limited RNA analysis of fixed cells must follow phenotypic analysis.
  • saponin used as a permeabilizing agent, was found to be a highly selective and efficient releasing agent for intracellular cytoplasmic RNA and other biomolecules, thereby obviating the need for cell lysis or homogenization.
  • This novel use of saponin as the RNA releasing agent of choice is a particularly advantageous component of the present invention.
  • Surfactants such as saponin have traditionally been used to examine the expression of intracellular antigens by permeabilization of the cell membrane allowing for incorporation of staining reagents while maintaining cell integrity.
  • RNA or DNA generally is done by solubilization or complete lysis of the cells with stronger surfactants, such as Triton X-100.
  • Saponin has heretofore not been used to study both expression of soluble intracellular antigens including RNA and phenotyping of individual cells or cell populations in the same specimen. Accordingly, methods allowing sequential phenotypic analysis as well as analysis of intact RNA and soluble proteins in the cytoplasm of the same cell specimen are highly desired and are the subject of this invention.
  • the present invention provides advantageous methods, apparatus, and kits for the rapid and efficient RNA profiling of all cells and especially targeted cells found in biological samples.
  • the present invention provides methods for allowing separate analysis of both phenotype and genotype. Phenotype is interrogated and profiled via antibody antigen protein and mass spectrometry profiling methods and comprehensive analysis of intact cytoplasmic RNA from the same cell or cell population. Genotyping of the sample genomic and mitochondrial DNA can be separately profiled by any means available to those skilled in the art. Similar to the amplification of the mRNA library, the respective genomic and mitochondrial libraries can be preamplified enabling numerous assays to be performed without loss of clinical sensitivity due to Multiple Displacement Amplification (MDA) technology enables the first effective whole genome amplification method. MDA is a rapid, reliable method of generating unlimited DNA from a few cells.
  • MDA Multiple Displacement Amplification
  • the invention described herein may be used effectively to isolate and characterize cell phenotype, such as cell surface antigens, intra-cytoplasmic antigens and any type of RNA, and genotype. Both phenotypic and genotypic analysis can be performed sequentially on the exact same sample. For example after cell surface analysis and RNA harvesting, the remaining intact nuclei and mitochondria can be analyzed downstream by all standard RNA (mt RNA, hRNA), DNA and protein based analysis techniques such as S1 nuclease, ribonuclease protection, RT-PCR, SAGE, DD-RT-PCR, microarray cDNA hybridization, ISH, FISH, SNP, all RNA and all genomic-based PCR techniques and any protein analysis systems.
  • mt RNA, hRNA DNA and protein based analysis techniques
  • S1 nuclease ribonuclease protection
  • RT-PCR ribonuclease protection
  • SAGE DD-RT-PCR
  • cancer is an organ specific disease when confined to its early stages. The disease becomes systemic by the time it is first detected using methods currently available. Accordingly, evidence to suggest the presence of tumor cells in the circulation would provide a first line detection mechanism that could either replace, or function in conjunction with other tests such as mammography or measurements of prostate specific antigen.
  • cellular phenotype protein and RNA
  • genotype By analyzing cellular phenotype (protein and RNA) and genotype through specific markers for these cells, the organ origin of such cells may readily be determined, e.g., breast, prostate, colon, lung, ovarian or other non- hematopoietic cancers.
  • RNA, and genome can be analyzed, especially where no clinical signs of a tumor are available, it will be possible to identify the presence of a specific tumor as well as the organ of origin.
  • these profiles define cell function, they also indicate what the most appropriate therapy type and course should be when used in cancer cell detection. Further in monitoring cases where there is no detectable evidence of circulating tumor cells as with post operative surgery or other successful therapies, it may be possible to determine from a further clinical study whether further treatment is necessary.
  • one embodiment of the invention includes the methods for isolating cytoplasmic biomolecules from a cell or population of cells, contacting the cell or cells with a permeabilization compound, and isolating the cytoplasmic biomolecule of interest from the cell while maintaining cell integrity for subsequent phenotypic and morphological analysis.
  • the targeted rare event in this invention refers to the expression of any biomaterial indicative, at least in part, to a known rare event. Accordingly, hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemical substances, nucleic acid molecules (such as RNA and/or DNA) and bioparticles such as cells, apoptotic bodies, cell debris, nuclei, mitochondria, viruses, bacteria, and the like would be included in the embodiment of this invention.
  • the fluid sample includes, without limitation, cell-containing bodily fluids, peripheral blood, bone marrow, urine, saliva, sputum, semen, tissue homogenates, nipple aspirates, and any other source of rare cells that is obtainable from a human subject.
  • Cytoplasmic biomolecules includes cellular target molecules of interest such as, but not limited to, protein, polypeptides, glycoprotein, oligosaccharide, lipids, electrolytes, RNA, DNA and the like, that is located in the cytoplasmic compartment of a cell.
  • target molecules of interest such as, but not limited to, protein, polypeptides, glycoprotein, oligosaccharide, lipids, electrolytes, RNA, DNA and the like.
  • the cytoplasmic biomolecules Upon contacting a cell with a permeabilization compound and subsequent cell separation, the cytoplasmic biomolecules are present in the supernatant for downstream analysis. All soluble cytoplasmic biomolecules, for example, the entire cytoplasmic RNA library or target components capable of traversing the membrane pores can be isolated and analyzed.
  • the focus is on the analysis of transcribed mRNA and translated proteins, for example in CTC, as indicators of oncogenic transformations of interest in the management of cancer diagnosis and therapy.
  • Membrane biomolecules includes any extracellular, intra-membrane, or intracellular domain molecule of interest that is associated with or imbedded in the cell membranes including, but not limited to, the outer cell membrane, nuclear membrane, mitochondrial and other cellular organelle membranes.
  • the targeted membrane biomolecules are normally not solubilized or removed from the membrane, i.e. the membrane biomolecules remain associated with the permeabilized cell.
  • Membrane biomolecules include, but are not limited to, proteins, glycoproteins, lipids, carbohydrates, nucleic acids and combinations thereof, that are associated with the cellular membrane, including those exposed on the external or extracellular surface of the outer membrane as well as those that are present on the internal surface of the outer membrane, and those proteins associated with the nuclear, mitochondrial and all other intracellular organelle membranes.
  • Membrane biomolecules also include cytoskeletal proteins.
  • Gene refers to the process of identifying intracellular genetic materials, such as DNA, that store internally coded inheritable instructions for constructing and controlling all aspects of cell life and death.
  • Phenotype or “phenotyping” is defined as classifying a cell on the basis of observable outward structural elements and the production thereof (i.e. including the intermediate RNA). These include topology, morphology and other surface characteristics, all of which result from internally coded genotypic information which are incorporated into the methods of the present invention.
  • RNA isolation techniques involving complete lysis of, at least, all cell structures except for nuclei and mitochondria in the presence of NP-40, usually by disintegration of all cell structures during chaotropic salt treatment and/or mechanical cellular homogenization.
  • Morphologic or morphology in reference to cell structure is used as customarily defined, pertaining to cell and nuclear topology and surface characteristics including intracellular or surface markers or epitopes permitting staining with histochemical reagents or interaction with detectably labeled binding partners such as antibodies.
  • morphology shall include the entire field of "morphometry" defined as: quantitative measure of chromatin distribution within the nucleus.
  • genomic and proteomic are used as conventionally defined. "Functional” is herein used as an adjective for an empirically detectable biological characteristic or property of a cell such as “functional cellomic” which more broadly encompasses both genomic and proteomic as well as other target categories including, but not limited to, "glyconomic” for carbohydrates and “lipidomic” for cellular lipids.
  • the resultant cell characteristics provide profiles permitting differentiation of normal from transformed cells.
  • Contacting means bringing together, either directly or indirectly, a compound or reagent into physical proximity of a cell.
  • the cell and/or compounds can be present in any number of buffers, salts, solutions, etc. Contacting includes, for example, placing the reagent solution into a tube, microtiter plate, microarray, cell culture flask, or the like, for containing the cell(s).
  • the microtiter plate and microarray formats further permit multiplexed assays for simultaneously analyzing a multiplicity of cellular target compounds or components including, but not limited to, nucleic acids and proteins.
  • Permeabilization compound, reagent, or composition means any reagent that forms small pores in the cell membranes, comprising the lipid-cholesterol bilayer, while maintaining sufficient membrane, cytoplasmic and nuclear structure such that subsequent phenotypic analysis can be carried out on the permeabilized cell(s).
  • saponin is a known "pore-forming" compound that complexes with cell membrane components thereby forming numerous trans-membrane pores of about 8 nm size in the cell wall or membrane, thus allowing outward diffusion of small soluble cytosolic constituents, such as enzymes, proteins, glycoproteins, globulins, electrolytes, and the like, and internal equilibration with extracellular reagent components, such as electrolytes.
  • Magnetic beads are magnetically labeled nanoparticles or microparticles also having covalently attached binding reagents (e.g. antibodies) with substantially selective affinity for surface markers or epitopes on cells, thereby achieving selective capture of magnetically labeled cells when exposed to a magnetic field such as generated in high gradient magnetic separation system (HGMS).
  • binding reagents e.g. antibodies
  • HGMS high gradient magnetic separation system
  • Preferred gene expression targets for identifying tissue of origin, diagnosis, prognosis, therapy target characterization and monitoring include but are not limited to cells derived from cancers of the breast, prostate, lung, colon, ovary, kidney, bladder, and the like for the purpose of detection and monitoring of sensitive or resistant genes expressing markers such as mammoglobin 1 (MGB1), mammoglobin 2 (MGB2), Prolactin inducible protein (PIP), carcinoembryonic antigen (CEA), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), glandular kallikrein 2 (hK2), androgen receptor (AR), prostasin, Hespin (HPN), DD3, Her-2/Neu, BCL2, epidermal growth factor receptor (EGFR), tyrosine kinase- type receptor (HER2), thymidylate synthetase (TS), vascular endothelial growth factor VEGF, pancreatic mucin (Mud), gu
  • circulating epithelial cells can be enriched relative to leukocytes to the extent of at least 2,500 fold to around 10,000 fold.
  • Immunomagnetic selection of circulating epithelial cells in blood is followed by a nucleotide analysis embodied in this invention. The enrichment is only one example of many methods known in the art for selecting specific populations of cells to be used in the embodiment of this invention.
  • a method of releasing intact cytoplasmic total RNA and mRNA from these cells, thereby isolating and purifying them, was unexpectedly and surprisingly discovered during conventional permeabilization of cells with saponin prior to staining and immunostaining, thereby enabling sequential or parallel analysis of both cytoplasmic RNA and intracellular antigen phenotyping and DNA genotyping on the exact same cell, population of cells, or specimen.
  • Permeabilization can be accomplished under this criteria using 1 of 3 types of general surfactants or detergents: pore forming reagents, like saponin, or saponin fractions such as QS-21 , escins, digitionin, cardenolides, etc. All of these agents increase membrane porosity and release small soluble intracellular components.
  • Another group of agents are surfactants. These agents have a relatively high hydrophilic-lipophilic balance to permeate the membrane without lysis. Other, more lytic surfactants with a lower hydrophilic-lipophilic balance, would release RNA, but tend to solubize the membrane.
  • polyoxyethylene sorbitans commercially known as Tween 20, 40, or 80
  • NP-40 nonylphenoxy polyethoxy ethanol
  • SDS t-octyl phenoxy ethoxy ethanol
  • cytoplasmic RNA and other RNA such as mtRNA and hnRNA
  • cell surface as well as soluble intracellular antigens
  • cell organelles such as mitochondria
  • the remaining indexed nuclei can then be analyzed downstream by all standard RNA, DNA, and protein based analysis techniques.
  • RNA, DNA, and protein based analysis techniques include all types of cDNA, RNA and protein microarrays for profile analyses, mass spectrometry, fluorescent in situ hybridization (FISH), single nucleotide polymorphism (SNP), all genomic- based amplification techniques such as PCR and the like, microsatellite analysis, restriction fragment length polymorphism (RFLP, ALFP), SAGE, DD- RT-PCR, and the like.
  • FISH fluorescent in situ hybridization
  • SNP single nucleotide polymorphism
  • RFLP restriction fragment length polymorphism
  • ALFP restriction fragment length polymorphism
  • Such analyses can be conducted on as few as 1-10 RNA molecules for each and any RNA sequence type, but preferably on tens of thousands up to millions copies of targets to enable detection of subtle alterations in cellular translation or transcription profiles as indicators of disease states in a clinical setting.
  • Other functional cell profiles of releasable and non-releasable cellular components can similarly be generated by analyzing the two fractions by conventional microarray, HPLC, electrophoretic methods including the high- resolution 2D electrophoresis method, or antibody array profiling.
  • Permeabilization compounds of this invention include, but are not limited to, saponins, a class of natural products constructed of cholesterol-like aglycones or genins (triterpenes or steroids not bearing any carbohydrate moieties) linked to fatty acids and one or more carbohydrates, which disperse readily in water to form globular micelles, the active species in pore formation.
  • suitable pore formers polyoxyethylene sorbitans (commercially known as Tween 20, 40, or 80), nonylphenoxy polyethoxy ethanol (NP-40), and t-octyl phenoxy ethoxy ethanol, have a high HLB (hydrophilic-lipophilic balance) numbers which must be used at sufficiently low concentrations to minimize undesirable solubilization of cellular components and membrane lysis.
  • the concentration range of the permeabilization compound is about 0.01-0.5% (w/v) when using saponin containing about 10% sapogenins.
  • a preferred permeabilization compound is saponin (Sigma Catalog Number S-7900).
  • Saponins from other sources and of higher purities may also be used, for example, saponin of about 20-25% purity as sapogenin (Sigma S-4521) and a highly purified saponin, QS-21 , of about 99% purity available from Aquila Biopharmaceuticals, Framingham, MA.
  • Other usable compound are alpha-escin and beta-escin (Sigma E-1378), both derived from horse chestnuts.
  • the permeabilization compound may be present in a composition, such as phosphate buffered solution, that also comprises antimicrobial agents such as, for example, sodium azide, Proclin 300 (Rohm&Haas, Philadelphia, PA), and the like.
  • ImmunipermTM Another preferred permeabilizing agent is ImmunipermTM, which by itself releases about 50% of the cytoplasmic RNA (85% of all RNA in the cell) with no affect on the nuclear or mitochondrial nucleotide pools. The remaining 50% of the total cellular RNA and all DNA in fixed cells can be released with a releasing cocktail comprising SDS, protease, and a formaldehyde scavenging agent, which composition constitutes one embodiment of this invention. While the exact mode of action of the individual cocktail components is unknown, it is speculated that the SDS serves to solubilize intracellular RNA and DNA crosslinked to structural intracellular proteins thereby enabling more efficient proteolysis and release of formaldehyde cross-linked nucleic acids.
  • novel formaldehyde scavenging reagents exemplified by, but not limited to, hydroxylamine, carboxymethoxylamine, hydrazine, acethydrazide and other hydrazides, or hydrazine derivatives, and amines such as tris, were found to increase the amount and "quality" of the released nucleic acids, where quality is measured by increased amplification rates and yields.
  • the two fractions released with Immuniperm and the releasing cocktail can be individually analyzed or pooled prior to analysis.
  • any surfactant or protease (or combination thereof) with or without added formaldehyde scavenger, capable of releasing cellular nucleotide stores and maintain a suitable morphology for concurrent analysis, would be included within the scope of the present invention.
  • the present invention enables extraction and isolation of greater than 90% of the intact cytoplasmic total RNA and mRNA from cells treated with a permeabilization agent, such as saponin, that permeabilizes the cell membrane while maintaining cell integrity.
  • a permeabilization agent such as saponin
  • RNA polymerase promoters based linear amplification methods employing T7, SP6, or T3 promoters, flowcytometry, microarrays and in Cell Spotter® or CellTracks systems (both manufactured by Immunicon Corp, PA) can be used to directly validate, complement and expand the expression profiles and enhance the information obtained therefrom.
  • permeabilized cells are treated with a cross-linking agent to maintain morphological, antigen and nucleotide integrity as stated above.
  • Cyto-ChexTM, StabilCyteTM and TRANSfixTM are examples of three commercially available stabilizers that have shown utility in stabilizing blood cells in blood specimens for extended time periods. These stabilizers are optimized to maintain cell size (mainly by minimizing shrinking) and to preserve antigens on cell surfaces, primarily as determined by flowcytometry.
  • the intended applications generally involve direct analyses and do not require extensive manipulation of the sample or enrichment of particular cell populations.
  • the circulating tumor cells, or other rare target cells, isolated and detected in this invention comprise and are defined as pathological abnormal or rare cells present at very low frequencies, thus requiring substantial enrichment prior to detection.
  • Cyto-ChexTM stabilizer can be used as a cell stabilizer and, as proven in application of the present invention, an aldehyde releasing fixative of intracellular RNA resulting in the formation of macromolecular complexes with intracellular proteins.
  • fixation preferably with a formaldehyde donor such as CytochexTM, was essential for retaining and protecting RNA during subsequent sample processing, and that total or optimal release of fully functional RNA required saponin in combination with the above-cited release cocktail.
  • the ideal "stabilizer” or “preservative” is defined as a composition capable of rapidly preserving target cells of interest present in a biological specimen, while minimizing the formation of interfering aggregates and/or cellular debris in the biological specimen, which in any way could impede the isolation, detection, and enumeration of targets cells, and their differentiation from non-target cells.
  • a stabilizing agent when combined with an anti-coagulating agent, a stabilizing agent should not counteract the anti- coagulating agent's performance. Conversely, the anti-coagulating agent should not interfere with the performance of the stabilizing agent.
  • the disclosed stabilizers also serve a third function of fixing, and thereby stabilizing, permeabilized cells, wherein the expressions "permeabilized” or “permeabilization” and “fixing”, “fixed” or “fixation” are used as conventionally defined in cell biology.
  • stabilizing agents herein implies using these agents at appropriate concentrations or amounts, which would be readily apparent to one skilled in cell biology, where the concentration or amount is effective to stabilize the target cells without causing damage.
  • concentration or amount is effective to stabilize the target cells without causing damage.
  • One using the compositions, methods, and apparatus of this invention for the purpose of preserving rare cells would obviously not use them in ways to damage or destroy these same rare cells, and would therefore inherently select appropriate concentrations or amounts.
  • the formaldehyde donor imidazolidinyl urea has been found to be effective at a preferred concentration of 0.1-10%, more preferably at 0.5-5% and most preferably at about 1-3% of the volume of said specimen.
  • An additional agent, such as polyethylene glycol has also been found to be effective in stabilizing cells, when added at a preferred concentration of about 0.1%-5%. The use of such agents is described in PCT/US02/26867, and is incorporated by reference herein.
  • a surprising aspect of the present invention is that intracellular RNA as part of the macromolecular complex can be recovered amplifiable and in nearly quantitative yields from cells previously treated with a cell stabilizer and fixative.
  • Full release of cross-linked RNA requires saponin in combination with enzymatic digestion in the presence of a lytic detergent and a formaldehyde scavenger.
  • a formaldehyde scavenger for example, proteinase K, V8 proteinase, pronase digestion of Cyto-ChexTM treated cells results in complete recovery or full- length comprehensively analyzable RNA.
  • the presence of a formaldehyde scavenger as disclosed in the present invention was found to further improve RNA recoveries.
  • target cells such as circulating cancer cells or fetal cells can be assayed by efficiently isolating them from other non-target cells, purifying their nucleic acids, and then amplifying the target(s) of interest for microarray analysis.
  • isolation of cytoplasmic biomolecules is achieved by first separating the permeabilized cell from the permeabilization compound through centrifugation or immunomagnetic bead enrichment. The cytoplasmic biomolecule mixture is then present in the supernatant. Isolation of cytoplasmic biomolecules can be achieved by capture with magnetic beads. For example if the cytoplasmic biomolecules are mRNA, oligo(dT) affixed to magnetic beads or nonmagnetic supports can be used to capture and thereby separate the mRNA from the cells with or without centrifugation. If the cytoplasmic biomolecules are proteins, antibodies that are able to bind to the particular protein can be used, wherein the antibodies can be affixed to magnetic beads or nonmagnetic supports.
  • Immunomagnetic enrichment reagents and devices for separating cells and biomolecules are available from several manufactures including but not limited to Immunicon Corp. (Huntingdon Valley, PA), Dynal (New Hyde Park, NY) and Miltenyi Biotec Inc. (Auburn, CA).
  • the cells can be prokaryotic, such as bacterial cells, or eukaryotic, such as mammalian cells, and are most preferable of human origin.
  • the cells are carcinoma or tumor cells.
  • Carcinomas of preferred interest include, but are not limited to, those derived from breast, prostate, lung, colon, and ovarian tissues, and the like, as found in tissue sections or in body fluids, for example, as circulating tumor cells in blood and bone marrow.
  • Methods are disclosed for preparing a cell for cytoplasmic and or whole cell biomolecule analysis and membrane biomolecule analysis sequentially on the exact same sample, collectively defined as either functional genomics or functional proteomics for analyses of nucleic acids or proteins, respectively. As stated above, such analyses have not heretofore been possible on the same cell(s) prior to the methods of this invention.
  • the cells are contacted with a permeabilization compound to release cytoplasmic biomolecules, as described above, without altering structural biomolecules and membrane biomolecules.
  • the methods of analyzing a cytoplasmic biomolecule from a cell sample and analyzing a membrane biomolecule from the same cell sample are provided after the cells are contacted with a permeabilization compound, stabilized, and a cytoplasmic biomolecule recovered as described above.
  • a cytoplasmic biomolecule can be isolated and analyzed concurrently or consecutively with an associated biomolecule.
  • kits for isolating cytosolic or whole cellular RNA, in particular, mRNA.
  • the kits may include a permeabilization compound and RNA extraction reagents or hybridization probes for RNA isolation and detection, such as for example, oligo(dT) or gene-specific sequences or random (degenerate) oligonucleotides of various lengths.
  • the kits can also include antibodies that bind to proteins associated with cells, such as antibodies that bind to membrane biomolecules.
  • the antibodies and probes can be enzymatically labeled, fluorescently labeled, or radiolabeled to allow detection.
  • the antibodies and probes can also be attached to, for example, magnetic beads or the like, to facilitate separation.
  • Cytoplasmic biomolecule analysis includes any type of analysis or assay that involves a biomolecule isolated from the cytoplasm of a cell. Cytoplasmic biomolecule analysis further includes, but is not limited to, functional genomic expression profiling including, but not limited to, mRNA profiling, protein expression profiling, reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, nucleotide or amino acid sequence analysis, serial analysis of gene expression SAGE, competitive genomic hybridization (CGH), electrophoresis, 2-D electrophoresis, mass spectrometry by MALDI or SELDI, gas chromatography, liquid chromatography, nuclear magnetic resonance, infrared, atomic adsorption, and the like.
  • functional genomic expression profiling including, but not limited to, mRNA profiling, protein expression profiling, reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, nucleotide or amino acid sequence analysis, serial analysis of gene expression SAGE, competitive genomic hybridization (CGH), electrophores
  • Sequence analysis at the nucleotide or amino acid level, can indicate and identify the presence of a mutation in a protein, DNA/cDNA, or mRNA sequence.
  • an original gene or protein profile analysis may indicate the presence of an oncogene in a transformed or tumor cell.
  • Subsequent analysis after appropriate cancer therapy may show lower tumor burdens during remission or indicate regression as a result of further mutations of the oncogene and emergence of drug-resistant or more aggressive tumor cells.
  • Membrane biomolecule analysis includes any type of analysis or assay that involves a biomolecule bound to or associated with a cellular membrane within a cell, i.e. extra-cellular and intracellular biomolecules or markers.
  • Appropriate analytical methods include, but are not limited to, flowcytometry, enzyme-linked immunosorbant assay, morphological staining, cell sorting, and the like.
  • Permeabilized cells can be sorted by, for example, fluorescence activated cell sorting (FACS) techniques based upon the expression of a particular detectable protein. Cell sorting techniques are well known to the skilled artisan and have been used to simply count detectably labeled cells, for example, in cancer diagnosis. Permeabilized cells can also be classified on the basis of expression of a particular protein, e.g.
  • Membrane biomolecule analysis can also be done on downstream membrane fractions followed by analysis, including, but not limited to protein expression profiling. Western blotting, amino acid sequence analysis, mass spectrometry, gas chromatography, liquid chromatography, nuclear magnetic resonance, infrared, atomic adsorption, surface plasma resonance (SPR) and any other technique suitable for analysis of membrane components.
  • Western blotting amino acid sequence analysis, mass spectrometry, gas chromatography, liquid chromatography, nuclear magnetic resonance, infrared, atomic adsorption, surface plasma resonance (SPR) and any other technique suitable for analysis of membrane components.
  • genomic analyses or assays can be performed on the genetic material that is retained within a permeabilized cell.
  • genomic DNA nuclear (hnRNA), mitochondrial (mtRNA) and any other RNA or DNA harbored by an organelle that remains bound or fixed within the cell upon permeabilization of a cell can be assessed.
  • the types of analyses described above for cytoplasmic biomolecules can be performed for genomic DNA, hnRNA, and mtRNA using methods or assays including, but not limited to, in situ hybridization, polymerase chain reaction, differential display PCR, arbitrarily primed PCR, microsatellite analysis, single nucleotide polymorphisms (SNP), competitive genomic hybridization (CGH), restriction fragment length polymorphism analysis, nuclear and mitochondrial transcript run-on assays, and in vitro protein translation assays.
  • the permeabilized cells must either be exposed to the releasing cocktail of the present invention, completely lysed, or further fractionated by conventional means well known to the skilled artisan.
  • combinations of proteinase and nucleophiles can be used to reverse and remove macromolecular complexes containing the nucleic acids of interest, liberating RNA and DNA nucleic acid components.
  • cell organelles retained upon permeabilization can be subsequently further fractionated and isolated for metabolic functional assays of, for instance, mitochondria and the like.
  • another embodiment of the present invention provides methods of separating nuclear or mitochondrial genetic material from cytosolic RNA.
  • Cells containing the nuclear or mitochondrial genetic material and cytosolic RNA are contacted with a permeabilization compound, as described above.
  • Nuclear or mitochondrial genetic material can be isolated by, for example, subsequent appropriate sub-cellular fractionation and complete cell/organelle lysis of the fractionated cellular material.
  • the resultant organelle specific components DNA, RNA, proteins, lipids, carbohydrates, etc.
  • Separation can also be accomplished using organelle-specific immunomagnetic beads, as described above.
  • the mRNA can be captured with oligo(dT)-magnetic beads that are ideally suited for automated downstream manipulation and comprehensive analysis similar to microarrays.
  • oligo(dT)-magnetic beads that are ideally suited for automated downstream manipulation and comprehensive analysis similar to microarrays.
  • minor changes are required in the current mRNA analysis protocols to generate both protein and mRNA profiles thus reducing the time and reagent requirements.
  • the corresponding intact cellular genomic DNA in the nuclei and mitochondria is still contained and accessible in the permeabilized cells and can be analyzed downstream by conventional methods for DNA, RNA and protein such as FISH, SNP, SAGE, DD-PCR, PCR, RFLP, RT-PCR, CGH, cDNA microarrays, mass spectrometry and protein arrays, etc.
  • Simultaneous multicomponent analysis strategies of DNA, RNA, protein, lipid, carbohydrate, and (precursors, metabolites, and co-factors thereof), for example, on large microarrays can thus be broadly applied to any eukaryotic cell, tissue sample or body fluid.
  • This type of cell expression profiling by means of multicellular component or combined with multiplexed (e.g. microarray) analyses is a cutting edge objective in technologies ranging from high-throughput screening of drug candidates to disease diagnosis and management.
  • kits for isolating cytosolic or whole cellular RNA, in particular, mRNA.
  • the kits may include a permeabilization compound and RNA extraction reagents or hybridization probes for RNA isolation and detection, such as for example, oligo dT or gene-specific sequences or random (degenerate) oligonucleotides of various lengths.
  • the kits can also include antibodies that bind to proteins associated with cells, such as antibodies that bind to membrane biomolecules.
  • the antibodies and probes can be enzymatically labeled, fluorescently labeled, or radiolabeled to allow detection.
  • the antibodies and probes can also be attached to, for example, magnetic beads or the like, to facilitate separation.
  • the intact library is then interrogated for the presence of any messages involved in identifying the presence of epithelial cells and/or confirming the presence of the tissue of origin of those epithelial cells.
  • all of the mRNA present in the sample must be analyzed for each particular gene of interest, each with the same sensitivity/selectivity as the other and with the ability to look at all the mRNA of interest at one time.
  • the signal-to-noise ratio in the sample would be impracticably low because of such problems as the white blood cell immunomagnetic carryover contamination in any given enriched sample.
  • a fluid sample enriched for a particular target population of cells by immunomagnetic selection there potentially could be approximately 10,000 white blood cells carried over with a target population of 1 to 10 cells.
  • the target cell(s) is expressing the rare event of interest, and would be masked by the nucleotides found in the white blood cells.
  • the excessive white blood cell derived background RNA noise coupled with the extremely rare copy level of the target mRNA results in a potential signal that may not be detected.
  • total RNA is pre-a plified by employing either a SP6, T3, or T7 RNA polymerase promoter-based in vitro linear pre-amplification method.
  • a typical example is T7 RNA polymerase (T7RNAP), promoter (T7RNAPP) and enzyme amplification system, but any equivalent system can be substituted by systems obvious to those skilled in the art.
  • T7RNAP T7 RNA polymerase
  • T7RNAPP promoter
  • enzyme amplification system any equivalent system can be substituted by systems obvious to those skilled in the art.
  • the linear pre-amplification of all messages increases the original mRNA library representation at least 1000 fold with minimal distortion of relative abundance of individual mRNA sequences within the RNA population.
  • the same pre-amplification process may also be known as transcript amplification, linear amplification, or in vitro amplification.
  • RNA polymerase creates antisense copies of the entire mRNA library (aRNA).
  • aRNA mRNA library
  • the T7 promoter oligonucleotide primer utilized as the first strand RT primer and a subsequent T7RNAP amplification primer, is composed of 67 bases having a 3'oligo(dT) portion containing a 5' T7 RNA polymerase promoter sequence having the following base pair order:
  • the pre-amplification reaction is completed by a reverse transcription reaction followed by randomly primed DNA polymerase dependant second strand synthesis and finally an overnight incubation with T7RNAP. Subsequently, a portion of this entire reaction mix is used in a PCR reaction analysis, which generates a specific single band amplicon with the appropriately designed gene specific primers (GSP's) of interest or any other appropriate RNA analysis method of choice.
  • GSP's gene specific primers
  • gene specific primers will depend upon the particular target sequence to be amplified and can be designed by any means known and accepted in the art.
  • gene specific primers are designed using the NCBI (National Center for Biotechnology Information) BLAST ® (Basic Local Alignment Search Tool) software and GenBank human cDNA sequence database.
  • the primers are optimized for annealing temperatures at about 55°C to 65°C and shown to produce only DNA-free, RT-PCR dependant single bands from complex mRNA libraries, which are known to be positive for particular mRNA.
  • the complex mRNA libraries are often extracted from normal and cancerous human tissues as well as in vitro cell lines.
  • the designed primers produce desired target sequence specific PCR bands that are all electrophoresed on agarose gels in order to compare design-predicted molecular weights with known standards. Calculations are completed using R f values determined on gel analysis software.
  • the amplicon sequences can be further sequence verified by direct sequencing, blot probing, restriction enzyme mapping, etc.
  • Second strand synthesis of the pre-amplified library is only within selected regions and could include from 1 to 1000 independent regions of interest for a single sample and still maintain the 100% sensitivity from the original library. Second strand synthesis is completed by selective amplification of only those genes of interest. Therefore, gene specific primers (GSP) are designed for second strand synthesis to include only the regions of interest. The regions would include for example, but not limited to, prostate specific antigen (PSA), PSM, CK19, EpCam, AR, HPN, F6, mamoglobin, and/or all the cytokeratins. GSP are designed to incorporate a universal primer on their tail end.
  • PSA prostate specific antigen
  • PSM PSM
  • CK19 EpCam
  • AR EpCam
  • HPN HPN
  • F6, mamoglobin and/or all the cytokeratins.
  • GSP are designed to incorporate a universal primer on their tail end.
  • part of the novel aspect of this invention is the use of the gene specific primers for only the second strand synthesis without the use of CAPswitch.TM.oligonucleotide, (U.S. Pat. No. 6,352,829).
  • the gene specific primers are designed to incorporate an arbitrary anchor sequence at their 5'ends which includes the CAPswitch oligonucleotide. So surprisingly with the invention herein disclosed, a universal portion of the primers does not include the CAPswitch moiety.
  • the length of the gene specific primers will typically range from about 15 to 30 nucleotides, while the universal primer portion will typically be about 15 in length.
  • Reverse transcription of a small portion of the T7 amplified antisense RNA (aRNA) library is performed using cycling conditions known in the art. All RT- PCR results are initially analyzed on 2% agarose gel containing ethidium bromide again according to procedures known in the art.
  • the product is then analyzed in an array format or by any electrophoresis format known in the art.
  • a universal PCR multigene amplification can be accomplished in a single tube, incorporating a set of gene specific primers (P1) for simultaneous reverse transcriptase in conjunction with the appropriate set of opposing primers (P2) for simultaneous second strand synthesis. Together, they define both (alpha and beta) termini and form a complete set of gene specific amplicons equaling a GSP multigene panel of interest.
  • the GSP1 and GSP2 priming for both gene specific first and second strand syntheses are conducted with the appropriate enzymes and under conditions of high primer-target annealing specificity, which are know to those skilled in the art.
  • Additional levels and approaches to achieving the appropriate primer specificity can be achieved by using proteins from natural recombination cellular repair mechanisms such as recA. Appropriate application of these repair systems in vitro will enable superior, even absolute, primer template specificity of formation.
  • the template criteria is either mRNA, or mRNA:cDNA heteroduplex, or double stranded duplex cDNA.
  • the innovative idea of utilizing a cell's natural repair mechanisms, as described, in the present application can be applied toward other gene specific primer methods such as the one described below for GSPs-RT subsets for signal to noise shifting enabling cDNA array analysis on rare cell events.
  • Each P1 and P2 primer in any one GSP multigene panel set of PCR primer contains a universal primer sequence at the 5' terminus which is common to all gene specific P1's and P2's (or just P1's and a separate universal sequence which is common to all P2's).
  • all GSP 1 and 2 are be removed from the desired double stranded cDNA amplicon panel set to eliminate their non-specific impact on down stream processes.
  • RNA-DNA oligo-primer hybrids could be used in place of DNA-Uracil and similarly be eliminated after first and/or second strand synthesis via DNase-free RNase treatment.
  • UNG degradation strategy will produce oligos much smaller than are capable of annealing under chosen PCR annealing temperatures.
  • the cDNA template mixture might also benefit from treatments with DNase-free RNases to eliminate all undesirable side reactions, possibly caused by high complexity RNA.
  • UNG treatment with an optional RNase treatment to eliminate all RNA
  • the only nucleic acids remaining are hybridized 1 st and complimentary 2 nd strands forming dsDNA duplexes, which now constitute the sample's available PCR templates.
  • non-UMP containing universal primers (1 or 2 max) are added for the follow-up PCR.
  • the net effect is the capturing of any desired set of mRNA (or DNA minus the RT) sequences with one or 2 PCR compatible high efficiency primers enabling quantitative RT- PCR multigene simultaneous amplification and subsequent analysis in a single tube. Since the primers are universal, they prime each GSP amplicon with the exact same efficiency, eliminating the confounding multiplex GSP primer performance problems.
  • Each GSP defined amplicon with a panel or set of amplicons can have a different predetermined fragment size enabling each GSP sequence to be resolved and identified by its unique Rf value in size-based analysis systems such as vertical and horizontal PAGE and agarose gel electrophoresis, capillary gel electrophoresis, SELDI, MALDI.cDNA arrays, etc.
  • size-based analysis systems such as vertical and horizontal PAGE and agarose gel electrophoresis, capillary gel electrophoresis, SELDI, MALDI.cDNA arrays, etc.
  • Preamplification adds the advantage of analyzing a single sample in up to 1000 different assays, thus many different panels of genes can be interrogated at different times on one sample. While not limited to any specific method, analysis of the universal PCR panels by cDNA array or capillary gel electrophoresis (CGE) is a preferred methodology.
  • CGE capillary gel electrophoresis
  • a critical feature differentiating the present invention from conventional technologies of the prior art is the improvement in signal to noise by selective amplification of rare target mRNA species, making this method a novel development over existing multivariate mRNA analysis.
  • Known multivariate analysis systems for example multiplex RT-PCR, can substantially change signal to noise, however the challenges of designing and optimizing meaningful multiplex systems has rendered them generally impractical especially for more than two target subsets in a reaction vessel.
  • This invention also utilizes the high signal to noise improvement to select representative transcripts, and amplifies in one reaction vial the entire set of target sequence(s) to be detected.
  • a set(s) of representative gene specific primers can be used to generate target gene subset(s) found in known disease states.
  • the representative set is will include at least two different target genes that are indicative of the disease state of interest.
  • the number of sets of gene specific primers will be determined by the disease state and the known characteristics that would define the disease state.
  • the supernatant obtained from ferrofluid selected unfixed cells that are permeabilized with Immuniperm, a phosphate buffered solution containing 0.05% saponin and 0.1% sodium azide was found to contain greater than 80% of the cellular total RNA residing in the cytoplasm of the cells.
  • the RNA isolated from this supernatant showed no evidence of degradation as judged by native and denaturing agarose gel electrophoresis and ethidium bromide staining.
  • This supernatant solution which is normally discarded after intracellular staining of the ferrofluid selected cells, was unexpectedly found to contain the RNA in an intact or undegraded full-length form thus providing an mRNA profile of the same cells that were also used for morphologic analysis.
  • Figure 2 illustrates these findings showing that total RNA release occurs in less than one minute and that about 95% of the cytoplasmic total RNA can be readily and reproducibly isolated.
  • RNA isolated from cells of the breast cancer cell line SKBR3 using the conventional process i.e. isolation by a commercially available phenol based RNA lysis buffer Trizol® Reagent (Gibco BRL, Gaithersburg, MD, Cat # 10296), completely lyses and homogenizes the entire cellular structures, thereby also resulting in the liberation of the genomic and mitochondrial DNA, and the cytoplasmic, mitochondrial and nuclear RNA.
  • duplicate tubes containing about 250,000 cells of the breast cancer cell line SKBR3 were first immunomagnetically enriched and then incubated in the absence (PBS only) and presence of Immuniperm (+IP) for 15 minutes at room temperature.
  • the Immuniperm treated permeabilized cells were then separated by centrifugation for 5 minutes at 800x g RCF as a visible permeabilized cell pellet.
  • the Immuniperm supernatant fraction containing all the cytoplasmic soluble components was transferred to a second tube.
  • Total RNA isolated from whole untreated cells, the Immuniperm treated permeabilized cell pellets, and the Immuniperm treated cell supernatant fractions were isolated using the RNeasy® (Qiagen Inc., Valencia, CA) silica binding method.
  • the gel images in Figure 3 also show that the rRNA are full- length and have high integrity as evidenced by the relative ratios of the 4.4 kb ssRNA co-migrating 28S rRNA bands compared to the 2 kb ssRNA marker co-migrating with the 18S rRNA bands.
  • the observed relative ratios of 28S rRNA to 18S rRNA of approximately 2 are an excellent indication of mRNA integrity as was further demonstrated by Northern blotting this gel and probing it with oligo(dT) as shown in Figure 4.
  • Literature values for the percent of total RNA contributed from the nucleus range from 15 to 25%. The Immuniperm® treated cell fractions containing 20% RNA are thus consistent with the published nuclear contribution.
  • Immuniperm-based permeabilization was unexpectedly shown to provide complete separation of nuclear and cytoplasmic total RNA with nearly 100% of the cytosolic total RNA readily recoverable in the supernatants of the Immuniperm treated unfixed cells. Furthermore, the nuclear fraction of total RNA surprisingly was found to remain intact in the resultant permeabilized cell structure following Immuniperm treatment.
  • RNA portions of the RNA derived from the two Immuniperm® cell fractions were evaluated against whole cells by Northern blot transfer of the denatured RNA from the gel shown in Figure 3 to a positively charged nylon filter.
  • Oligo(dT) 25-mer probe was labeled with 32 P and poly nucleotide kinase then hybridized to the Northern blot.
  • Single- stranded RNA size ladders containing poly-A tails were used as markers enabling the formation of a molecular weight banding ladder for relative qualitative sizing of mRNA populations.
  • the oligo(dT) hybridization results in Figure 4 shows that no significant differences in size ranges were observed for the mRNA libraries between the three samples.
  • the release of Immuniperm-derived mRNA is not limited by transcript size since nearly 100% of the cytosolic mRNA is retrievable from the Immuniperm supernatant, and the integrity of rRNA 28S/18S is indicative of full retention of mRNA integrity.
  • FIG. 4 The Northern blot shown in Figure 4 was stripped and re-probed with nuclear specific precursor rRNA probe.
  • Figure 5 shows that Immuniperm treatment of cells achieves a complete separation of nuclear and cytosolic total RNA populations. Thus, the nuclear membrane structure remains intact during the Immuniperm treatment and the nucleus retains all its soluble components.
  • the Northern blot in Figure 5 was stripped and re-probed with a mitochondrion-specific 12s rRNA probe.
  • the results, shown in Figure 6, demonstrate that Immuniperm treatment of the cells achieves complete separation of mitochondrial and cytosolic RNA populations. Thus, the mitochondrial membrane structures remain intact during the Immuniperm treatment.
  • RNA stocks solutions which were used to generate the images in Figures 3-6, were further used to generate 32 P-labeled first strand cDNA libraries of equal masses and specific activities.
  • These three labeled first strand library probes were hybridized to obtain separate but identically prepared cDNA array dot blots as shown in Figure 7.
  • the objective was to evaluate the mRNA relative abundances represented in each Immuniperm derived cell fraction by comparing the relative proportions of each cDNA hybrid signal pattern for each imaging filter.
  • the randomly selected cDNA gene array identities on the template are shown in Figure 7.
  • g6 macrophage inhibitory cytokine 1
  • CK8 cytokeratin 8
  • CK18 cytokeratin 18
  • CK19 cytokeratin 19
  • EpCam epithelial cell adhesion molecule
  • uPA urokinase plasminogen activator.
  • the respective lengths of the transcripts in this cDNA array varies from 1 to 5 kb, again reinforcing the Northern blot finding that no size bias is seen in the release mRNA from Immuniperm treated cells.
  • the identical relative representation patterns in Figure 7 also unexpectedly demonstrate that the lmmuniperm®-derived mRNA is as effective a reverse transcriptase template for first strand synthesis as the whole cell mRNA derived by traditional methods.
  • Figure 8 shows the gel image of the cytosolic RNA obtained by treating about 770 PC-3 cells with Immuniperm.
  • the data show that Immuniperm- derived cytosolic mRNA from lower cell numbers give the same proportions of RNA as were demonstrated in Figures 3, 4, 5, 6, and 7. Therefore, Immuniperm-mediated cell release of cytosolic RNA is not cell number dependent.
  • Isolation of circulating tumor cells from peripheral blood followed by cell analysis by flowcytometry and gene expression analysis by RT-PCR can be performed as follows: EDTA-anticoagulated blood (7.5 ml) is transferred into a 15 ml conical tube and 6.5 ml of System Buffer (PBS also containing 0.05% sodium azide, Cat #7001 , Immunicon Corp., Huntingdon Valley, PA) is added. The tube is securely capped and mixed by inverting several times. The blood- buffer mixture is centrifuged at 800x g for 10 minutes at room temperature. The supernatant is carefully removed by aspiration taking care not to disturb the buffy coat layer. Some supernatant can be left in the tube. The aspirated supernatant can be discarded.
  • System Buffer PBS also containing 0.05% sodium azide
  • AB Buffer System Buffer containing streptavidin as a reversible aggregation reagent, Immunicon Corp., Huntingdon Valley, PA
  • VU/desthiobiotin EpCAM ferrofluid particles Immunomagnetic nanoparticles coupled to anti- EpCAM monoclonal antibody also conjugated to desthiobiotin for biotin- reversible aggregation with streptavidin, Immunicon Corp., Huntingdon Valley, PA
  • VU/desthiobiotin EpCAM ferrofluid particles Immunomagnetic nanoparticles coupled to anti- EpCAM monoclonal antibody also conjugated to desthiobiotin for biotin- reversible aggregation with streptavidin, Immunicon Corp., Huntingdon Valley, PA
  • the cell-buffer mixture is carefully aspirated using a Pasteur pipette and the aspirated supernatant is discarded.
  • the tube is removed from the separator and 3 ml of System Buffer is added.
  • the magnetically collected cells are resuspended by brief vortexing. The liquid should rise up the tube during vortexing so that cells near the top are washed down.
  • the uncapped tube is again placed in the QMS17 separator for 10 minutes and the supernatant is aspirated with a Pasteur pipette. The aspirated supernatant is discarded.
  • the magnetically collected cells are resuspended by vortexing in 200 ul of Immuniperm/RNase inhibitor (Permeabilization reagent, Immunicon Corp., Huntingdon Valley, PA) also containing RNase inhibitor, RNase OUT, Cat. # 10777019, Invitrogen, Rockville, MD).
  • the liquid should rise up the tube during vortexing so that all cells are washed down.
  • Antibodies such as, for example, monoclonal anti- cytokeratin antibody (C11-PE, 0.25 ug) (cocktail of antibodies recognizing cytokeratins 4, 5, 6, 8, 10, 13, 18 conjugated to R-Phycoerythrin; Immunicon Corp., Huntingdon Valley, PA) in a 25 ul volume and 10 ul of CD45 PerCP (Pan anti-leukocyte marker, Cat. # 347464, Becton Dickinson, San Jose, CA) or any other suitable antibodies can be added and mixed by vortexing. After 15 minutes of incubation, the sample is gently agitated by lightly tapping the bottom of the tube. The tube is returned to the QMS17 for 5 minutes. The supernatant is gently aspirated and the Immuniperm-RNA fraction transferred to an appropriately labeled tube.
  • C11-PE monoclonal anti- cytokeratin antibody
  • CD45 PerCP Pan anti-leukocyte marker, Cat. # 347464, Becton Dickinson, San Jose,
  • the cells from Example 2 are resuspended in 200 ul of CellFix (PBS based buffer containing biotin as a de-aggregation reagent and cell preservative components, Immunicon Corp., Huntingdon Valley, PA) and incubated for 5 minutes.
  • the sample is transferred to a 12 x 75 mm flow tube and 300 ul of PBS are combined, followed by the addition of the nucleic acid dye thioflavin T (Sigma # T3516, 10 ul) and about 10 ul of fluorescent beads (10,000 beads; Flow-Set Fluorospheres, Cat. # 6607007 Coulter, Miami, FL).
  • the sample is mixed by vortexing.
  • the fluorescent beads tube is mixed by vortexing before pipetting the beads.
  • the sample is then analyzed on a flowcytometer.
  • the poly(A)+ mRNA is isolated using magnetic oligo(dT) labeled beads (Dynabeads® mRNA Direct® Micro Kit, Dynal, Prod. # 610.21 , New Hyde Park, NY).
  • total RNA can be isolated by using any other appropriate means to those skilled in the art such as silica binding, polymer binding, and more traditional phenol extractions like Trizol® Reagent (GibcoBRL, Cat # 10296).
  • Genomic DNA is eliminated by treatment with DNase enzyme such as DNase I (GibcoBRL).
  • An enzyme mix composed of 2:1 of 10x DNase I (1 U/:1), 1 :1 of RNase inhibitor (cloned), 5:1 of dH 2 0, and 10:1 of RNA or control (250ng Genomic DNA) is prepared.
  • the enzyme mix is incubated at 37°C for 20 minutes.
  • the DNased RNA is re-purified by magnetic oligo(dT) labeled beads or Trizol® isolation and resuspended in 10:1 RNase-free water.
  • the activity of DNase enzyme is confirmed by running the control genomic DNA (+/- DNase treatment) on a 2% agarose gel with ethidium bromide staining.
  • Specific mRNA sequences can be amplified using rTth (Thermos thermophilis) RT-PCR.
  • a 40:1 volume of Master Mix is added to the sample tube containing 10:1 of DNased RNA and corresponding negative control tubes containing 10:1 of H 2 0.
  • PCR thermocycling is carried out for 40 cycles as follows: 50°C for 2 minutes (pre-PCR), 62°/65°C for 30 minutes (pre-PCR), 95°C for 1 minute (pre-PCR), 94°C for 15 seconds (PCR), 62°/65°C for 30 seconds (PCR), and 62°/65°C for 7 minutes (post-PCR).
  • the sample tube is immediately placed in a - 20°C block for 2 minutes. After completion, the sample tube is placed in a 4°C block until gel analysis will be performed.
  • a volume of 20:1 is run on a 2% agarose gel with ethidium bromide staining.
  • Qualitative and quantitative gene expression measurements of specific mRNA transcripts are made by examination of the gel image using a UV transilluminator and an alpha imager for the presence of the amplicon at the expected molecular weight.
  • the soluble proteins in this supernatant solution and the insoluble proteins remaining in or on the surface of the cells thus provide a means for determining the total protein expression profile or proteomics profile of the cells as well as the cellular morphology.
  • the fraction of total cytosolic soluble protein liberated from the cytoplasm due to Immuniperm treatment is determined relative to the total amount of protein liberated from a duplicate cell preparations treated with NP- 40, a surfactant that is the preferred reagent for total cytosol protein release from cells.
  • Both treated cell preparations are freed from membrane debris via centrifugation or magnetic separation prior to determination of total soluble proteins by conventional methods, such as the spectrophotometric Lowry and Bradford methods.
  • aliquots of the two sample preparations are electrophoresed in a 4 to 20% gradient SDS polyacrylamide gel to (a) determine the molecular weight cut-off for Immuniperm-derived cytosolic proteins and (b) compare the protein banding patterns and relative quantities of protein per band in the two preparations.
  • aliquots are further analyzed by 2D electrophoresis and conventionally stained or detectably labeled to provide "fingerprint" information on sizes and isoelectric points of the proteins in the two fractions based on the qualitative and quantitative spot patterns of identifiable and unidentified components.
  • the derived information generates proteomic expression profiles of the relative and absolute protein expression patterns in the cytosolic and total protein compartments of normal and transformed cell populations.
  • GSP Gene Specifically Primed
  • S/N Signal-to-Noise
  • GSP Gene Specifically Primed
  • Traditional RT-PCR or the more desirable quantitative versions can be applied however they are generally considered a poor use of individual samples as these samples yield very small amounts of starting material.
  • clinical sensitivity is compromised for multigene analysis.
  • unamplified mRNA/cDNA libraries can only be analyzed one time for only one gene without compromising clinical (and maximum technical) sensitivity. With individual samples being scarce, several higher throughput methods were developed.
  • RNAPA RNA polymerase amplification
  • This GSP subset RNAPA selection process is reduced to practice in this example using a model system that reflects typical WBC mRNA copy number ratios found in clinical samples (10ng total RNA in approximately 5000 WBC) / CTC (0.5ng total RNA in approximately 50 CTC).
  • the starting WBC mRNA total copy number proportional to the non-specific background noise was approximately 10 8 to 10 9 .
  • the starting total RNA/mRNA was subjected first to one round of amplification which increased proportionately all the mRNA species approximately equal as determined by real-time quantitative RT-PCR (Table 1). Subsequently, a 25ng aliquot of the first round amplified aRNA was subjected to a second round of GSP subset RNAPA, shifting the signal-to-noise of the 4 GSP targets as described below (Table 1).
  • RNAPA In the second round GSP RNAPA, a key selection step occurs during the single RT reaction forming simultaneous first strands only for a predetermined mRNA library subset of which the gene specific RT primers are included.
  • the subset of GSP RT primers were for the above 4 mRNA (PSA, PSM, AR, EpCAM).
  • GSP-RT selective first strand synthesis is followed by synthesis of the complimentary second strand using the appropriate DNA polymerase and oligo(dT) primer bearing a T7RNAP promoter, thus creating a selective set of double stranded DNA templates T7RNAP ready.
  • RNAPA enabled templates have been selected via GSP first and second strand synthesis.
  • all remaining RNA is degraded by exposing the second strand reaction mix with a cocktail of DNase-Free RNases.
  • any remaining single stranded RNA and any extraneous (non-poly U/ poly A dependent) single stranded cDNA which was formed during dT dependent second strand synthesis can be eliminated by single-strand-specific nucleases such as Mung Bean Nuclease.
  • double-stranded cDNA template subsets are purified by phenol extraction and/or silica binding.
  • RNAPA ready templates are RNAP amplified overnight to yield an approximately 1000 fold increase of only 4 genes of interest in S/N shifting over the other possible templates such as the F6 (alpha 1 globin sequence) which represents WBC mRNA derived noise.
  • Table 1 shows the results of real-time quantitative RT- PCR for these 4 genes of interest throughout the process including subsequent GSP-second round S/N shifting where F6 is defined as alpha 1 globin sequence found in this system to be highly abundant in WBC and not detectable in epithelial cells.
  • Cyto-ChexTM and other formaldehyde and formaldehyde-urea derivative based fixatives stabilize approximately 100% of full-length total RNA, mRNA and other nucleic acids in all cells found in whole blood when compared to matched non-fixed controls.
  • Intact RNA stabilized as macromolecular complex, changes its RNA chemical characteristics and is unaffected by current traditional cell lysis and chaotropic salt based RNA isolation methods such as phenol extraction, silica binding and oligo(dT) hybridization.
  • fixative recovered RNA are combined with an aRNA preamplification or universal PCR methods described in the present application for comprehensive analysis down stream or for general functional enablement of total and mRNA library.
  • Figure 9 demonstrates that Cyto-ChexTM performs like other aldehyde based fixatives. Upon fixative exposure for 24 hours, less than 1% of the mRNA and a disproportionate amount of 18S-rRNA are recoverable (approximately 10%). Even when extreme chaotropic salt denaturation chemistries are applied (i.e. GITC and Phenol, silica or (dT) hybridization, BRL's Trizol Reagent, Qiagen's RNA mini silica binding and Dynal's Dynabeads mRNA Direct oligo (dT) poly (A) + kits) recovery is extremely low.
  • extreme chaotropic salt denaturation chemistries i.e. GITC and Phenol, silica or (dT) hybridization
  • BRL's Trizol Reagent Qiagen's RNA mini silica binding
  • TransfixTM formulation used here achieves a 0.1% final concentration of paraformaldehyde fixative per unit volume blood.
  • Formaldehyde-urea derivatives in the presence of numerous macromolecular nucleophiles found in biological systems leads to an increase in the rate of dissociation of these derivatives.
  • Dissociation occurs in close proximity to biological nucleophile complexes, possibly regulatory proteins specifically associated with RNA that leads to covalent linkages. These linkages and associations are then removed and reversed by subsequent proteinase and stronger nucleophile treatment.
  • TransfixTM yields full-length high integrity mRNA libraries from 24hr stabilized whole blood cells demonstrates that all aldehyde based stabilizers will yield nucleic acids of similar high quality. Thus resulting in a reproducible yield of nucleic acids after preservation and recovery.
  • mRNA transcript size distribution proportions for each nucleophile are different even though none of the samples shows RNA degradation. This suggests that different types of mRNA sequences are retrievable (i.e. different types of formaldehyde modifications are reversed) by specific nucleophiles and incubation conditions.
  • the various enzymes used also show different proportions recovered ( Figure 11 , bottom of gel).
  • Figure 12A shows the resultant relative quality and quantity of aRNA from a single T7RNAP preamplification of the total RNA/mRNA isolated in triplicate of 10 and 20 SKBR3 cell spikes into 7.5ml peripheral blood after stabilization with Cyto-ChexTM for 24 hours at room temperature. Each of these replicates was then immunomagnetically enriched from which the cell lysate was treated with proteinase reversal conditions and followed by silica binding total RNA isolation. Normalized equivalents of aRNA were used in quantitative RT-PCR reactions for two specific genes, CK19 and EpCAM. The results of which are shown in Figures 12B and 12C.
  • Figures 12D and 12E shows the varying mRNA RT-template quality that is derived from the three different aldehyde based fixatives, Cyto-ChexTM StabilcyteTM and TransfixTM.
  • TransfixTM yields an mRNA template, which might be of a slightly lower RT quality than either Cyto-ChexTM or StabilcyteTM.
  • Figures 12A, 12B, 12C, 12D, and 12E confirm a reproducible, product viable, procedure for blood RNA sample preservation and comprehensive analysis of circulating epithelial cells, which are most likely cancer cells.
  • the high level of mRNA preservation is amenable to qualitative analysis that can detect the presence of single cell spiked into 7.5ml of blood for any mRNA that exists in that cell within at least 50 mRNA molecules/cell (i.e. only 50 copies / sample).
  • the rates and types of covalent fixations in whole blood vary according to the type of fixative.
  • the rates and types of covalent fixative reversal or recovery will vary according to the type or combination of proteinases and nucleophiles used.
  • the rate of fixation will be a critical issue for applications where the half-lives of mRNAs of interest are faster then the rate of fixation.
  • Both the forward fixation and the reversal recovery reactions (processes) can be optimized further yielding yet higher quality and quantity of RNA.
  • the current quality and quantity of the RNA stabilized and recovered is demonstrated here in blood to be far superior to anything previously shown.
  • EXAMPLE 8 ENRICHMENT AND ANALYSIS OF mRNA FROM CTC IN FRESH NON- FIXED BLOOD
  • Human blood was isolated from 9 patients with advanced hormone refractory prostate cancer (HRPC) and 13 healthy volunteers and assessed for gene expression mRNA specific to circulating epithelial cells.
  • HRPC advanced hormone refractory prostate cancer
  • Plasma samples were kept at room temperature and processed within 2-3 hours after collection unless otherwise indicated. 15 ml of blood were divided into 7.5 ml aliquots and transferred to disposable tubes with an internal diameter of 17 mm (Fisher Scientific) and centrifuged at 800 g for 10 min with the brake off. Phosphate buffered saline (PBS) with bovine serum albumin (BSA) was added to bring the volume up to 10 ml and the sample was mixed by inversion.
  • PBS Phosphate buffered saline
  • BSA bovine serum albumin
  • the Mab VU-1 D9 recognizing the epithelial cell adhesion molecule (EpCAM) is broadly reactive with tissue of epithelial cell origin and coupled to magnetic nanoparticles (ferrofluids, Immunicon, Huntingdon Valley, PA).
  • CA-EpCAM ferrofluid and a buffer containing streptavidin are then added to the sample to achieve this increase in the magnetic labeling of the cells.
  • Desthiobiotin on the CA-EpCAM ferrofluid is subsequently displaced by biotin, which is contained in the permeabilization buffer described below. Thereby reversing the cross linking between the CA-EpCAM ferrofluid particles.
  • the sample was immediately placed in a quadrupole magnetic separator for 10 min (QMS17, Immunicon). After 10 min, the tube was removed from the separator, inverted 5 times, and returned to the magnetic separator for an additional 10 min. This step was repeated once more and the tubes were returned to the separator for 20 min. After separation, the supernatant was aspirated and discarded. The tube was removed from the magnetic separator, resuspended with 3 ml of phosphate buffered saline (PBS) containing bovine serum albumin (BSA), and the fraction collected from the walls of the vessel.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • RNA or mRNA is isolated from the enriched cell population. Isolation can be accomplished by any means known in the art that is able to keep the mRNA intact and prevent degradation.
  • the enriched circulating tumor cells from duplicate blood samples were lysed in 100 ul of Trizol reagent (BRL) or 100 ul of RNA Extraction Buffer (ZYMO Research) and the vortex-homogenized sample was stored at -80°C until RNA was used. Homogenates were used to isolate total RNA according to manufacturers' instructions. Briefly, total RNA was treated with DNase I.
  • RNA integrity and mass was labeled with 32 P, phosphor imaged (Packard Cyclone) and analyzed to determine RNA integrity and mass. The remaining total RNA mass values (90%) from each sample were then designated as that sample's 7.5 ml blood donor equivalent of total RNA, 1.5% of which was calculated to be mRNA.
  • Flowcytometric analysis of leukocytes taken from human blood was assessed for gene expression in circulating epithelial cells. Isolated cells were prepared as described then ' resuspended in 200 ul permeabilization buffer containing biotin (Immunicon Corporation) to which monoclonal antibody (Mab)-fluorochrome conjugates were added at saturating conditions.
  • the monoclonal antibodies consisted of a Phycoerythrin (PE) conjugated anti- cytokeratin monoclonal antibody (Mab C1 1 ) recognizing cytokeratins 4,6,8,10,13, and 18 (Immunicon) and peridinin chlorohyll protein (PerCP)- labeled anti-CD45 (Hle-1 , BDIS, San Jose, CA).
  • PE Phycoerythrin
  • Mob C1 1 conjugated anti- cytokeratin monoclonal antibody
  • peridinin chlorohyll protein PerCP- labeled anti-CD45
  • the collected cells were resuspendened in 0.5 ml of PBS to which the nucleic acid dye used in Procount SystemTM was added (Procount, BDIS).
  • the nucleic acid dye used in Procount SystemTM was added (Procount, BDIS).
  • 10,000 fluorescent counting beads were added to the suspension to verify the analyzed sample volume (Flow-Set Fluorospheres, Coulter, Miami, FL)
  • Samples were analyzed on a FACSCalibur flowcytometer equipped with a 488 nm Argon ion laser (BDIS). Data acquisition was performed with CellQuestTM (BDIS) using a threshold on the fluorescence of the nucleic acid dye. The acquisition was halted after 8000 beads or 80% of the sample was analyzed. Muitiparameter data analysis was performed on the listmode data (Paint-A-GateTM, BDIS). Analysis criteria included size defined by forward light scatter, granularity defined by orthogonal light scatter.
  • the leukocytes carried over from the immunomagnetic selection ranged from 655 to 5,560 (median 4,350; mean 1 ,759). In HRPC patient samples, the leukocytes carried-over ranged from 813 to 92,000 (median 4,350; mean 12,300). Blood samples from healthy, non-cancer control group, 7 male and 6 female, showed no CTC whereas in the blood samples from HRPC showed a CTC range of 4-283 in 7.5 ml of blood.
  • RNA/aRNA mass Normalization of mRNA/aRNA mass was determined by first quantitating the total RNA mass isolated from each immunomagnetically enriched 7.5 ml blood sample volume. This was accomplished by Northern blotting 10% of each sample's total RNA, followed by 28S plus 18S radiolabeled oligo probe hybridization, and in parallel with known total RNA mass cell line standards. This was followed by phosphoimage quantitation (Cyclone, Packard Instruments).
  • CK19-cRNA in vitro transcribed RNA construct
  • This CK19-cRNA standard contained the 3'-most 800 bases of CK19 wild type mRNA sequence.
  • Standard CK19- cRNA curves covered a 1000 fold dynamic range were run in triplicates at 20,000; 2,000; 200; 100; 50; 25; and 12.5 copies each spiked into 2 ng total RNA isolated from Percoll-derived WBC.
  • Standard kinetics curves run for 40 cycles showed linear signal response plotting band intensity against RNA copy number between 13-200 copies CK19-cRNA transcript [see Figure 13A and 13B].
  • the external standard curve had a maximum CV of 27% for any standard analyzed in triplicate. For multivariate gene analysis, comparisons were made to CK19 external standard curves and relative gene expression levels 0 - 4 were assigned:
  • Figure 13B shows typical banding intensities that correspond to the approximate copy number of CK19 cRNA on the standard curve, and were used to assign relative gene expression levels 1 -A.
  • the CTC enumeration and gene transcript expression profiles were determined using 23 different PCR amplification products from Ep-CAM immunomagnetically enriched blood samples of 13 healthy donors and 9 HRPC patients. Microarrays were not effective for analyzing these types of samples due to the signal to noise incompatibility derived from the WBC which are nonspecifically carried over during the immunomagnetic enrichment process. The ratio of CTC specific signal to WBC carry-over noise in these samples ranges from 1 to 1000 CTC per 10 3 to 10 4 WBC. These microarray limitations were overcome by incorporating a 10,000 fold preamplification step using 90% of the entire mRNA library from each immunomagnetically enriched blood sample, followed by multigene RT-PCR analysis in place of the arrays.
  • This innovation provides enough starting material for several hundred individual PCR reactions to be performed with each 7.5 ml blood sample.
  • one is enabled to perform individual patient CTC multivariate RT-PCR profile analysis without compromising assay sensitivity or clinical sensitivity for each mRNA member of each CTC mRNA library.
  • RNA from each sample was reverse transcribed (RT) using a SMART PCR cDNA synthesis kit, but using the 67 base oligo(dT) primer described above. The reaction was incubated at 42°C for 90 min.
  • the entire 10 ul RT was transferred into a 50 ul PCR reaction using the Advantage cDNA PCR kit (Clontech) and subject to PCR with the P1 -SMART primer and P2-T7 18 base primer: (5'-TCTAGTCGACGGCCAGTGAATT-3') using a PE-9600 and thermal cycling program; 95°C for 1 min., 10 cycles of 95°C for 15 sec, 65°C for 1 sec, 68°C for 6 min.; followed by 20 min at 72°C.
  • the entire PCR reaction volume was loaded on a Sephadex G-50 Quick Spin (TE) column (Roche Diagnostics) and the eluate was generated according to the manufacturer's instructions.
  • RNA polymerase transcript amplification reactions that produced representative libraries of aRNA were assembled using AmpliScribe kit (Epicenter Technologies) according to manufacturer's instructions in a 20 ul volume and incubated at 37°C for 6-12 hours. Repeating the Trizol isolation procedure further cleaned up the RNA transcription reaction.
  • RNA size standards, RNA mass standards, and one tenth of the transcription reaction products from each sample were formamide denatured at 65°C for 15 min., loaded on 2% agarose gel, run for 15 min at 5 volts/cm, and post-stained with SYBR GoldTM (Molecular Probes) for one hour prior to gel image densitometry using AlphalmagerTM (Alpha Innotech Corp.). The mass of each transcript library was determined.
  • Gene specific primers were designed as describe. All primer sets were designed to amplify specific gene target cDNA within the 3'-most 500 bases
  • Table 1 shows the primer pairs for each amplicon analyzed by relative RT-PCR. Forward primer P1 is shown as the upper sequence in the respective primer pair. Reverse primer is the lower sequence. All sequences are written 5'- 3'.
  • NKX3A NM_006167 284 GGAAGTTCAGCCATCAGAAGTAC 246
  • PIP J03460 226 CAGAACTGTGCAAATTGCAGCCGTC 201
  • TROP2 X77753 334 TGTTGCTACTCTGGTGTGTCCCAAG 245
  • RT Reverse transcription
  • PCR curves were generated from each single PCR reaction tube by aliquoting 15 ul at 31 , 35, and 40 cycles during the thermal cycling program: 95°C for 1 min, and 31 ,35, and 40 cycles of 95°C for 1 sec, 65°C for 1 sec, 72°C for 1 min; followed by 20 min at 72°C using PE-9600 thermalcycler (or thermocycler??).
  • Each amplicon within each PCR batch included a cell line cDNA amplification positive control, and a master mixed PCR reagent amplification negative control that contained all components except for the cDNA sample. All RT-PCR results were analyzed on a 2% agarose gel containing ethidium bromide in parallel with BRL low mass molecular weight and spot densitometry analysis with AlphaEaseTM software version 5.04.
  • the gene survey goal was to identify mRNA expression profiles in CTC with the highest clinical specificity and sensitivity for detecting epithelial cells.
  • gene expression levels found in the WBC was evaluated from enrichment of healthy donors. The selection of gene candidates was based on known literature data that showed broad epithelial specific expression levels. Identification of candidates that were negative in WBC would enable CTC profiling for categorizations/characterization on three basic histological levels, epithelial origin ( Figure 14A), tumor/organ of origin ( Figure 14B), and tumor therapeutic target characterization (Figure 14C).
  • Figure 14A shows epithelial marker CK19 is 100% specific in the system of the present invention for HRPC samples, where 0/13 healthy donors were CK19 positive compared to 18/23 (78%). HRPC patient samples scored CK19 positive. CD5 and TROP2 are also 100% specific. However, their poor sensitivity of 1/23 (4%) and 2/23 (9%) respectively may not add sufficient profile value to justify their use. Of the genes that are not 100% specific (EpCAM, Muc-1 , and MIC-1), a WBC background threshold can be applied between levels 1 and 2, above which all patient derived signals greater than one can be considered true positives.
  • RNA from breast and prostate cancer cell lines SKBR-3 and LNCaP America Type Culture Collection, Manassas, VA
  • Trizol reagent Trizol reagent (Life Technologies Inc.) according to manufacturer's instructions.
  • each dilution was spiked into 2 ng of total RNA from Percoll-derived WBC.
  • RT-PCR external CK19 standard curve determination was run in parallel with SKBR-3 dilution curve, resulting in about 1 ,000 wild type CK19 mRNA copies present per SKBR-3 cell equivalent.
  • the CK19 characterized SKBR-3 dilution curve was used as one of two types of specific transcripts to model the lower limit sensitivity and reproducibility of the total process of this T7-based mRNA library amplification system.
  • the second transcript was an exogenous Lambda DNA based construct (Walker Biotech Publication) producing a 1.2 kb polyA(30) transcript.
  • the curve for WBC's spiked with SKBR-3 cells was assayed in triplicate at each of the 2000, 200 and 20 CK19 mRNA copy levels.
  • Lambda curves were spiked in triplicate into 2 ng total RNA from Percoll-derived WBC at the 500, 50, and 5 copy levels.
  • this lower limit of detection starting with only 50 mRNA transcripts of any one sequence, was reproduced in a subsequent cell line spike model where all six sequence types (PSA, PSM, AR, HPN CK19 and EpCam) were serially diluted to known copy levels prior to aRNA and quantitative RT-PCR analysis (Example 6).
  • Total RNA libraries were proportionately amplified using one round of the modified T7 method of the present invention yielding aRNA libraries with an average increase above the original mRNA mass of 10,000 fold. This is based on the original mRNA level estimation of 1.5% of the determined total RNA mass.
  • the transcript amplification process resulted in libraries with a medium transcript length of 600 bases, which range between 550-800 bases. Individual transcript sizes within each library ranged from 300-3000 bases.
  • Individual aRNA libraries were randomly primed for RT, from which a multigene panel of individual PCR reactions was performed using 10 donor equivalents of aRNA/cDNA.
  • Total RNA quantities from carried-over WBC's in healthy non-cancer samples ranged from 0.8-11.12 ng (mean 3.5 ng).
  • Total RNA quantities from HRPC patient samples ranged from 0.8-35.12 ng (mean 7.2 ng). All total RNA samples subsequently produced aRNA libraries of masses directly proportional to the starting total RNA values.
  • a Northern blot of 10% of each sample's total RNA was hybridized with 28S plus 18S radiolabeled oligo probes in parallel with a known mass of total RNA from cell line standards which was followed by phosphoimage for quantity and quality determinations. Quality was assessed by ratio of 28S over 18S quantities where SKBR-3 cell line standard was averaged 1.55 (range 1.50-1.64), for the enriched samples, 13 healthy donors averaged 1.36 (range 1.16-1.60), HRPC averaged of 1.10 (ranged 0.57-1.80).
  • T7 pre-amplification step allows the analysis of just a few copies of the transcript wit up to 1000 different individual gene specific RT- PCR reactions.
  • T7 pre-amplification of representative mRNA libraries effectively removes the major restriction of limited sample mRNA mass.
  • This same pre-amplification can be applied to non-CTC RNA. Indeed, there are numerous sources of RNA in a given blood sample, and some of these non- CTC RNA transcript will provide valuable information.
  • RNA derived from the plasma blood fraction obtained during the ferrofluid enrichment process can be coupled with the T7 pre-amplification process described in the above examples for enriched CTC.
  • the ferrofluid enrichment process initially separates out the blood plasma fraction of each sample. Typically, this fraction has been discarded as the CTC are enriched.
  • Plasma- and serum- derived mRNA and DNA have recently been shown in the literature to provide valuable cancer expression (phenotype) and genotype (DNA analysis) data.
  • Plasma-derived mRNA and DNA are isolated by traditional molecular biology methods for downstream analysis. Since mRNA is readily available from plasma, and has been demonstrated to provide valuable RT-PCR data, these same RNA transcripts can be more comprehensively profiled using the modified T7 amplification procedure describe herein.
  • CTC-independent and/or CTC- complimentary mRNA expression profiles can be generated with the same profiling procedures for CTC by using the RNA from the plasma-derived fraction of each sample.
  • the T7 based expression profiling approach can be applied to the above described enrichment process, allowing analysis of the CTC- depleted fraction can be useful for differentiating the contributions of the WBC expression profile, which is non-specifically carried over during enrichment, and the contributions form the CTC-specific profile. This can be accomplished by differential pattern comparison and subsequent subtractions, providing an additional mechanism for correctly identifying CTC during analysis.
  • the CTC-depleted profiles themselves will provide valuable patient-specific information regarding response and sensitivities to particular therapies.

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Abstract

L'invention concerne un dosage biologique hautement sensible, qui utilise un procédé pour l'amplification, à amorçage spécifique de gènes, de bibliothèques d'ARNm provenant de cellules rares et de transcrits rares trouvés dans le sang. Le dosage biologique selon l'invention permet de détecter de l'ARNm rare (10 copies/cellule) trouvé dans 1 à 10 cellules isolées par enrichissement immunomagnétique. Le dosage biologique selon l'invention constitue une amélioration par rapport aux PCR multiplex et permet la détection efficace de séquences de codage rares pour des cellules carcinomateuses circulantes dans le sang. L'invention concerne également des procédés permettant le profilage de cellules isolées dans des tissus ou des fluides corporels et pouvant servir de compléments au diagnostic clinique de divers carcinomes, incluant la détection précoce et la classification des cellules tumorales circulantes. La surveillance de profils d'acides nucléiques et de protéines de cellules, dans des formats conventionnels ou bien dans des jeux ordonnés de micro-échantillons, facilite la gestion de l'intervention thérapeutique, incluant la mise en place de la thérapie, la surveillance de la réponse à la thérapie, la confirmation de la rémission et la détection d'une régression.
PCT/US2002/034570 2001-10-26 2002-10-28 Analyse a parametres multiples d'acides nucleiques complets et de caracteristiques morphologiques sur le meme echantillon WO2003035895A2 (fr)

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IL16135702A IL161357A0 (en) 2001-10-26 2002-10-28 Multiparameter analysis of comprehensive nucleic acids and morphological features on the same sample
KR10-2004-7006181A KR20040047971A (ko) 2001-10-26 2002-10-28 동일 샘플상에서의 광범위 핵산 및 이의 형태학적 특질에대한 다중 매개변수 분석법
EP02795565A EP1438419A2 (fr) 2001-10-26 2002-10-28 Analyse a parametres multiples d'acides nucleiques complets et de caracteristiques morphologiques sur le meme echantillon
BRPI0213520-5A BR0213520A (pt) 2001-10-26 2002-10-28 método para diagnosticar a gravidade de uma doença em um indivìduo de teste, e, kit de teste para triagem de uma amostra de paciente quanto à presença de células cancerosas circulantes
CA002465288A CA2465288A1 (fr) 2001-10-26 2002-10-28 Analyse a parametres multiples d'acides nucleiques complets et de caracteristiques morphologiques sur le meme echantillon

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BR0213520A (pt) 2006-05-23
WO2003035894A3 (fr) 2003-12-04
AU2002360305A1 (en) 2003-05-06
CA2465274A1 (fr) 2003-05-01
WO2003035894A2 (fr) 2003-05-01
CA2465288A1 (fr) 2003-05-01
JP2005507997A (ja) 2005-03-24
IL161357A0 (en) 2004-09-27
KR20040047971A (ko) 2004-06-05
CN1610752A (zh) 2005-04-27
EP1438419A2 (fr) 2004-07-21
WO2003035895A3 (fr) 2004-01-08
WO2003035894A8 (fr) 2004-06-03
EP1438398A2 (fr) 2004-07-21

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