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WO2000008208A2 - Transcription inverse, amplification, et leurs amorces - Google Patents

Transcription inverse, amplification, et leurs amorces Download PDF

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Publication number
WO2000008208A2
WO2000008208A2 PCT/GB1999/002579 GB9902579W WO0008208A2 WO 2000008208 A2 WO2000008208 A2 WO 2000008208A2 GB 9902579 W GB9902579 W GB 9902579W WO 0008208 A2 WO0008208 A2 WO 0008208A2
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Prior art keywords
primer
cdna
amplification
primers
species
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PCT/GB1999/002579
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English (en)
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WO2000008208A3 (fr
Inventor
Thomas Charles Freeman
Peter John Richardson
Alistair K. Dixon
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Medical Research Council
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Priority to AU52943/99A priority Critical patent/AU5294399A/en
Publication of WO2000008208A2 publication Critical patent/WO2000008208A2/fr
Publication of WO2000008208A3 publication Critical patent/WO2000008208A3/fr

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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

Definitions

  • the present invention relates to processes for the reverse transcription of mRNA to provide cDNA. Additionally, the invention relates to processes involving reverse transcription of mRNA and concomitant or subsequent amplification of cDNA.
  • the mRNA samples are typically those obtainable from cell or tissue samples of organisms, such as biopsy or blood samples, for example.
  • the invention relates to processes for reverse transcription or reverse transcription and > amplification of a population of mRNA obtainable from a single cell.
  • the invention concerns the field of analysis of gene expression (i.e. "expression profiling") of cells and tissues, even down to level of a single cell.
  • the invention also relates to polynucleotide primers adapted for the performance of reverse transcription on population of mRNA species with concomitant or subsequent amplification procedures.
  • DNA sequence information resulting from genome and expressed sequence tag (EST) sequencing projects is expected to provide the basis for furthering understanding of the control and mode of action of individual gene products.
  • expression profiling is regarded as playing a role in the functional characterisation of newly identified genes (Lander, E S (1996), Science, 274:536-539 and Strachan, T (1997), Nature Genetics, 16:126-132).
  • Many tissues, such as the immune and nervous systems are composed of highly heterogeneous cell populations.
  • a key factor in understanding their physiology, and the role of specific gene products expressed within them, is to examine gene usage in the context of this cellular diversity. In the past, methods such as Northern blotting and nuclease protection assays were employed to study gene expression.
  • si tu hybridization techniques provide detailed information about the cellular expression pattern of a gene in intact tissue, be it whole-mounts or tissue sections, the technique is relatively laborious and unable to analyse multiple transcripts in a single preparation.
  • PCR polymerase chain reaction
  • Nested PCR has been used successfully in many scientific laboratories and it relies on two sequential amplification steps, both targeted to the genes of interest. This means, therefore that in any one cell sample the expression of only a few genes (of up to 3 gene families) can be examined.
  • the technique does not ensure that all members of cDNA populations in a complex mixture are amplified, nor that all the amplicons are of similar sizes.
  • the technique suffers from the drawback that only a few gene families can be examined at a time. This is wholly unsatisfactory from the point of view of expression profiling.
  • This technique is suited to the analysis of the expression of no more than about 3 genes in any cell
  • the technique is useful for simultaneous expression screening of a large number of dead cells in fixed tissue slices
  • si tu hybridization usually involves the use of radiolabels which are inconvenient and the technique as a whole ' is quite unsuitable for expression profiling of living cells.
  • This technique used by a number of research groups employs terminal deoxynucleotide transferase to attach a unique priming site on to the 3' end of a first strand cDNA following reverse transcription. Following PCR of the cDNA with the unique priming site, the expression profile of a number genes from a single cell can be analysed.
  • the technique has a number of drawbacks. First, there is the need to use homopolymeric PCR primers capable of annealing to sites in a DNA sequence. Also, there is an unequal amplification of cDNA because unequal lengths of the cDNA transcripts are amplified. The amplification efficiency is low. The initial PCR reactions for the different transcripts operate at vastly different efficiencies, and so bias the procedure in favour of shorter gene transcripts.
  • a primer sequence is ligated to the 3' end of cDNA to provide a second amplification primer site.
  • this technique suffers from the same problems as the cDNA tailing technique referred to above. PCR of the different cDNA species in the reverse transcribed sample takes place at different levels of efficiency, depending on the length of the cDNA molecule being amplified. Additionally, the ligation reactions can be difficult to control with multiple priming sites being ligated.
  • the present inventors have sought to develop a more straightforward, reproducible and reliable cDNA amplification procedure for small mRNA samples wherein expression profiling can be conducted, thereby avoiding the various problems outlined above.
  • the present invention provides a process of reverse transcribing mRNA species present in a sample from an organism comprising:
  • the invention provides a process of reverse transcribing and amplifying mRNA species present in a sample from an organism comprising:
  • the particular sequence of the non-heel portion of each second primer in the second heeled primer population is such that at least one second heeled primer from the population is capable of hybridizing to each reverse transcribed first strand cDNA species.
  • the number and identity of individual second heeled primers which hybridize thereto is expected to be different but this is not an expectation which excludes the possibility of similarities in hybridizations with cDNAs arising.
  • their hybridization with the first cDNA strands is possible at a multiplicity of sufficiently complementary sites along the lengths of the cDNA strands .
  • the particular temperatures, enzymes and reagents (other than the first primer) used in the process of reverse transcription may be those already known in the art.
  • a “heeled” primer will be readily understood in the art to be a primer comprising a hybridizing portion and a non-hybridizing portion, wherein the non- hybridizing portion represents the "heel" of the primer.
  • the second primer is actually a population of individual primer species.
  • first strand cDNA population is contacted with the second primer population under appropriate hybridizing conditions then because of the selection of nucleotide sequences amongst the second primers, each cDNA species will hybridize with at least one second primer, second cDNA strand synthesis then proceeds in a 5 ' to 3 ' direction from the hybridized second primer.
  • second strand cDNA synthesis proceeds in its 5' to 3' direction from the second primer whose 3 ' end is not obstructed along the first strand cDNA template by any other second primer hybridized thereto.
  • a multiplicity of second primers hybridize to the first strand cDNA template
  • the 3' end portions of mRNA molecules are generally more diverse in their sequences.
  • a net result of the reverse transcription process of the invention is that there appears to be a bias towards a more uniform length of cDNA molecule. This in turn impacts on any subsequent amplification procedure.
  • the amplification is PCR for example then more uniform length cDNA molecules lend themselves more to amplification than a population of cDNA molecules less uniform in length.
  • the processes of the invention generate cDNA molecules highly representative of the spectrum of mRNA molecules in a sample.
  • the element of bias towards more uniform length cDNA molecules ensures that even relatively low abundance mRNA species are transcribed, and optionally amplified, to the same level of efficiency as more abundant mRNA species.
  • a much better qualitative profile of expressed gene sequences in samples can be achieved than was hitherto possible.
  • the degree of sensitivity of the processes of this invention can of course be varied by modifying the numbers and sequences of the second primer population species thereby modifying the frequency with which the second primers hybridize to the first strand cDNA templates .
  • the amplification of the cDNA species resulting from the reverse transcription preferably employs a third primer comprising at least a part of the heel portion of the first heeled primer and a fourth primer comprising at least part of the heel portion of the second heeled primer. Further amplification may be advantageous where subsequent analysis of cDNA species involves less sensitive detection means or where a larger sample is required for analysis by methods which require larger quantities of cDNA material.
  • the second heeled primer population may comprise primers differing by up to five nucleotide bases the population preferably comprising a number of primers in the range 1000 to 100,000 primers, more preferably in the range 1024 to 65536 primers.
  • the primers of the second heeled primer population preferably each comprise a random sequence of nucleotides in the range of 5 to 8 nucleotides 3' to the heel and a further sequence of at least 5 nucleotides contiguous 3' therewith. As will be appreciated, where there are 5 random nucleotides (which is preferred) there will be 4 5 (i.e. 1024) possible pentamer sequences.
  • the further sequence of nucleotides may be selected by sequence analysis of known sequences so as to promote the ability of the second heeled primer as a whole to hybridize to the transcribed cDNA species.
  • the sequence analysis can be carried out through databases of DNA or RNA sequences.
  • the known sequences of the organism of interest are preferably consulted.
  • the further sequence of nucleotides preferably comprises a number of nucleotides in the range 2 to 10 nucleotides.
  • the further sequence of nucleotides may comprise a number of nucleotides equivalent to the number of nucleotides in the random sequence of nucleotides.
  • the further nucleotide sequence of the second heeled primers is preferably constant throughout the population of these primers and it is selected so as to stabilise the primers and to ensure optimal efficiency of hybridization to target first strand cDNA species.
  • the second heeled primer from the population of second primers preferably hybridises on average once in every lkb portion of first strand cDNA species. This has been found to provide a relatively efficient and uniform reverse transcription and optionally amplification of mRNAs in a sample.
  • a particularly preferred further sequence of nucleotides in the second primers is:
  • N is independently selected from C G T or A.
  • the heel portion of the first and second heeled primers are preferably selected so that they lack the ability to hybridise to mRNA or first strand cDNA respectively.
  • the heel portions are selected by an analysis of known nucleotide sequence information.
  • the heel portions preferably comprise sequences absent from the mRNA species in the sample, although the heel portions may simply comprise sequences absent from the genome of the organism from which the sample is taken.
  • the heel portions preferably comprise a number of nucleotides in the range 15 to 50, more preferably 18 to 22 nucleotides although somewhat fewer or somewhat more nucleotides may be acceptable.
  • the first heeled primer is preferably an anchored primer comprising an oligo (dT) sequence.
  • anchored primers are already well known in this art. For example, provision of a few non-T bases at the 5' end of the primer ensures that hybridization of the primer occurs at the 5 1 end of the mRNA poly A tail.
  • the fourth primer is preferably the heel of the second heeled primer, or at least a portion thereof.
  • the third primer is preferably the heel of the first heeled primer, or at least a portion thereof.
  • the third primer may be the same as the first heeled primer and this can be advantageous in reducing the numbers of reagents needed to perform the processes of the invention.
  • the frequency with which individual second primer population species hybridize along a given length of nucleic acid may be adjusted by employing suitable hybridizing conditions.
  • the hybridization conditions are of limited stringency so that the random sequences of oligonucleotides in the second primers have a significant effect on whether hybridization occurs or not.
  • the degree of stringency of hybridizing conditions and the number of contiguous random bases in the second primers may be varied according to routine trial and error in order to achieve a desired frequency of hybridization of second primer species along a given length of nucleic acid material.
  • the amplification of the resulting cDNA species preferably comprises more than one round of amplification cycles.
  • each further round of amplification comprises addition of further second and third primers and amplification reagents, optionally a fourth primer as well.
  • Each round of amplification may comprise 5 to 45 cycles, more preferably 10 to 40 cycles.
  • the first round of amplification may comprise a lesser number of cycles than any further rounds of amplification.
  • the amplification process is preferably PCR although modified PCR procedures or other compatible amplification procedures may be used.
  • Preferred PCR cycles comprise X ⁇ °C for Yi min; then X 2 °C for Y 2 min; then X 3 °C extension for Y 3 minute; then Y 4 min extension, wherein X l r >X 3 >X 2 and Y 4 >Y 3 >Y 2 >Y ⁇ .
  • Xi is in the range 90 to 94 ⁇ C
  • X 2 is in the range 45 to 70°C
  • X 3 is in the range 65 to 75°C and Yi
  • Y 2 , Y 3 are in the range 15 seconds to 4 minutes and Y 4 is in the range 2.5 to 20 minutes.
  • the sample from an organism will preferably include or be derived from tissue or cells.
  • the sample may be comprised of whole cells, possibly comprising a single cell type, even comprising just a single cell.
  • the samples are composed of the cytoplasm of cells (same cell type or mixture) or more preferably the cytoplasm of a single cell.
  • Biopsy samples may provide useful sources of sample material ranging from a few grams to a few micrograms of tissue/cell material.
  • the cytoplasm may be obtained by lysis or aspiration of a cell or cells and such cell may be obtained by fluorescence activated cell sorting (FACS) .
  • FACS fluorescence activated cell sorting
  • the ' processes of the invention are sufficiently reliable, sensitive and efficient that substantially all mRNA species in a sample are reverse transcribed and optionally amplified to approximately the same degree. A more accurate and reliable picture of gene expression can be obtained for a cell sample.
  • the processes of the invention permit single cell gene profiling.
  • the -invention provides a method of reverse transcribing expressed gene sequences in a sample from an organism comprising reverse transcribing the mRNA in the sample using a first primer to produce first strand cDNA species, synthesising second cDNA strands using a population of second primers, wherein at least one second primer in the population hybridises to a given first strand cDNA species.
  • This method may further comprise the amplification of the resulting double stranded cDNA.
  • Preferred or alternative versions of this method may comprise one or more of the further features of the other processes of the invention as hereinbefore described.
  • the invention provides a polynucleotide primer for reverse transcription of mRNA species comprising an oligo (dT) sequence and 5' thereto a polynucleotide heel sequence, wherein the heel sequence is substantially incapable of hybridisation to the mRNA species.
  • the primer is preferably an anchored primer and may comprise the further features as hereinbefore described.
  • the invention provides a polynucleotide primer population for synthesis of second strand cDNA species from first strand cDNA species, wherein at least one primer in the population is capable of hybridising to a given first strand of cDNA. At least one primer in this primer population is preferably capable of hybridising approximately at least once in any given lkb of first strand cDNA.
  • the primers may further comprise one or more additional features of such primers as hereinbefore described.
  • the invention provides polynucleotide primers for amplification of cDNA comprising a reverse transcription primer (ie the first primer) as hereinbefore described and a primer comprising at least a portion of the heel portion of the second primer population as hereinbefore described.
  • the invention provides the use of a polynucleotide comprising an oligo (dT) sequence and a heel sequence 5' thereto for the reverse transcription of mRNA species in a sample.
  • a polynucleotide may further comprise one or more of the characteristics of the first primer as hereinbefore described.
  • the invention provides the use of a polynucleotide primer population as hereinbefore defined for the synthesis of second strand cDNA from a population of first strand cDNA species.
  • the invention provides a cDNA library preparation obtainable by a process or method as hereinbefore defined, said library comprising substantially all cDNA species corresponding to genes expressed by a single cell, cell type or tissue.
  • the invention provides a kit for the production of cDNA from mRNA m a sample from an organism comprising a primer of the fourth aspect of the invention and a primer population of the fifth aspect of the invention.
  • the kit may further comprise at least one further primer for achieving amplification of the cDNA.
  • Particularly preferred kits comprise a primer pair for amplification of the cDNA.
  • the invention thus provides a rapid, robust and reproducible procedure, called Three Prime End
  • TPEA Amplification
  • TPEA-PCR PCR
  • An important advantage of TPEA-PCR is the relative ease of performing the method. Other known procedures are generally time consuming and complex, involving DNA purification and precipitation from one step to another.
  • the present cDNA amplification technique however, can be carried out in a single tube with a need for only limited manual intervention. This therefore makes it possible to amplify large numbers of samples relatively easily.
  • the ability to then analyse the expression of many genes of unrelated sequence, both at high and low abundance, in samples taken from as little as a single cell, will potentially allow it to be used in high throughput screening systems.
  • the invention can be used to analyse gene expression in samples as small as just a single cell ( Figure 4), or in much larger samples such as 100 cells ( Figure 2).
  • Amplification from a single cell currently provides enough material for approximately 40 gene specific PCR reactions. Whilst this is already an improvement over existing protocols, it should theoretically be possible to improve the efficiency of the TPEA reaction to provide far higher yields of 3' cDNA product. This would then not only allow the number of gene-specific PCR reactions performed on each sample to be increased, but more importantly allow the procedure to be linked to other analysis procedures.
  • TPEA-PCR Three Prime End Amplification optionally with PCR
  • TPEA Three Prime End Amplification optionally with PCR
  • the 3' region of mRNA is amplified arbitrarily by PCR using a combination of primers.
  • the amplified cDNA which represents the most diverse region of gene sequence, can then be analysed by a second round of PCR using gene-specific primers.
  • Using the invention it is possible to analyse the expression of, for example, up to 40 genes (20 in duplicate) in single human lymphoblastoma cells.
  • the method is also suited to the analysis of genes expressed at low levels in small populations of cells, eg expression of the adenosine A 2a receptor in cholinergic neurons of the rat striatum.
  • Sequence diversity between genes is at its greatest in the 3 ' untranslated region and this region provides the most unique target for gene-specific assays; this is especially important when wishing to differentiate between closely related members of a gene family.
  • the procedures of the invention preferentially amplifies this portion of the mRNA sequences.
  • cDNA synthesis by reverse transcriptase is initiated by an anchored oligo-dT priming so that the 3' region of all genes is represented in the resulting single-stranded cDNA.
  • a 5 '-specific heel may be incorporated into this primer for use in the subsequent amplification procedure.
  • Second- strand synthesis it is desireable that about 1 kb from the 3' end of each gene is selected and amplified by PCR Assuming a completely random length of nucleotide sequence, it would be expected that a given 5 base sequence would appear every 1024 bases (4 5 ) , even though some nucleotide sequences are more common than others (Lopez-Nieto & Nigam (1996) Nature Biotech 1_4: 857-861) . A pentameric sequence is preferentially selected so that the primer initiates second-strand synthesis in an arbitrary manner within 1 kb of the 3' end of the mRNA. A search of 30 gene sequences reveals that at least one copy of this 5 base sequence was present in this region of each gene.
  • each DNA strand contains a specific priming site 5' and 3' to the region of interest, thus allowing amplification of the intervening sequence.
  • the majority of the mRNA species represented in the first-strand cDNA pool before amplification, as detected by conventional RT-PCR are also detectable after amplification .
  • the invention should permit the analysis of gene expression in samples obtained from small samples of tissue or single cells. In so doing, it should allow the utilisation of the wealth of new sequence data now available, to further understanding of disease processes and the cellular physiology of complex issues .
  • TPEA-PCR A major utility of TPEA-PCR will be in sampling single cells in tissues and in culture conditions to conduct detailed studies of temporal gene expression, changes in gene expression in response to growth conditions or environmental insults, and in identifying hitherto undetected gene activity associated with particular cellular events. Apart from the study of cells to understand their innate biology and responses, new approaches to toxicology profiling are promised as well as means to molecularly classify rare cell types.
  • the invention has many possible applications including 1. Making a single cell cDNA libraries for subsequent detailed analysis of gene expression, and the discovery of novel genes.
  • FIG. 1 shows a schematic illustration of the process of TPEA-PCR. Details of the protocol are given in the Examples, as are the particular sequences of the anchored oligo (dT) primer and the partially degenerate second strand primer.
  • Figure 2 shows cDNA amplification. cDNA was prepared from varying numbers of sorted lymphoblastoma cells, and analysed by RT-PCR for the expression of 8 "housekeeping" genes, before (left) and after (right) TPEA-PCR. After cDNA amplification, expression of all 8 genes assessed could be detected after carrying out gene specific PCR on only 5% of the amplified cDNA generated from a single cell.
  • Genes assayed RPL21 (riboprotein L21) , RP27a (riboprotein 27a), RPL28 (riboprotein L28), RPS5 (riboprotein S5), HSKPQZ7 (housekeeping protein) , ACTB (Cytoplasmic beta-actin) , G-3-PDH (Glyceraldehyde-3-phosphate dehydrogenase) and EF1 (Elongation factor 1) .
  • Figure 3 shows multiple gene expression analysis in single lymphoblastoma cells.
  • Four cells (1-4) were lysed, reverse transcribed, the cDNA amplified and gene specific PCR performed on the product.
  • CD2, SI and intron primer pairs serve as negative controls to check for genomic contamination of the samples. Eleven of the other genes are expressed in all four cells in duplicate, while JUND, EF1 CDC25B and CD19 expression was not consistent between cells.
  • RPL5 riboprotein L5
  • RPL21 riboprotein L21
  • RP27a riboprotein 27a
  • RPL28 riboprotein L28
  • RPS5 riboprotein S5
  • RPS9 riboprotein S9
  • RPSI0 riboprotein SI0
  • RPS29 riboprotein S29
  • HSKPQZ7 Housekeeping protein
  • ACTB Cytoplasmic beta-Actin
  • G-3-PDH Glyceraldehyde-3-phosphate dehydrogenase
  • EF1 Elongation factor 1
  • JUND JUND
  • CDC25B cell cycle factor CDC25b
  • IGM immunoglobulin IgM
  • SI the intestine-specific enzyme, sucrase-isomaltase
  • Figure 4 shows adenosine 2a receptor expression in striatal cholinergic interneurons .
  • Panel a shows an infrared video image of a rat striatal cholinergic interneuron during electrophysiological characterisation and panel b, after -aspiration of cytoplasm.
  • the expression of four housekeeping genes, the transmitter synthesising enzymes choline acetyltransferase (found only in cholinergic neurons) and glutamic acid decarboxylase (found in GABAergic, medium spiny neurons) , three tachykinin (NK) receptors and the adenosine A 2a receptor was assessed in twenty six striatal cholinergic neurons. Two representative neurons are shown, neuron 1 expresses the A 2a receptor, neuron 2 does not, while the expression of the other genes tested are the same in both.
  • TPEA-PCR assay was performed on lymphoblastoma cells in the G0/G1 phase of the cell cycle. Groups of 100, 10 and single cells were flow sorted into wells containing lysis buffer and the mRNA reverse transcribed. A proportion of the sorted cells then underwent 3' end amplification, as described hereinafter.
  • Figure 1 shows a schematic summary of the TPEA-PCR procedure. Gene-specific PCR assays for 8 'housekeeping' genes were carried out on lymphoblastoma cDNA, before and after cDNA amplification. Following reverse transcription only, the expression of each of the housekeeping genes could be detected when cDNA generated from between 1 and 100 cells was used in each PCR assay as shown in Figure 2.
  • Epstein Barr virus transformed lymphoblastoid cell line (HRC575, ECACC, Porton Down, UK) was maintained in log phase growth in RPM1 1640 medium supplemented with 16% foetal calf serum, 2mM L-glutamine and penicillin-streptomycin (100 U/ml and 100 mg/ml respectively) .
  • Cells at approximately 10 8 per ml were stained with the bixbenzimadazole dye Hoechsht 33342 (Sigma, Poole, UK) at 1 ⁇ g/ml for 30 minutes at 37°C.
  • Cells were sorted by using the Autoclone attachment of a Coulter Elite ESP flow cytometer, 300 mW of all lines UV from a Coherent 306 laser and by using single drop and complete abort sorting settings. Time of flight, forward and right angle scatter, and Hoechst fluorescence peak and area measurements were used to ensure the sorting of single cells. The accuracy of sorting (both spatial and numerical) was tested by sorting ' single fluorescent beads (DNA Check, Coulter Corp) into 96 well plates and viewing the plates on a fluorescence microscope.
  • RT Reverse Transcription
  • cDNA Amplification Lymphoblastoma cells were FACS sorted into 96 well plates containing 7 ⁇ l of freshly prepared lysis buffer (50 mM Tris-HC (pH 8.3), 75 mM KC1, 3mM MgCl 2 , 5mM NP-40 (Sigma) and 1.5 units of RNase inhibitor (Pharmacia, Milton Keynes, UK) .
  • This buffer leaves the nucleus intact (Jena et al (1996) , J Immunol Methods, 190:199-213).
  • RNA reverse transcriptase (Gibco BRL, Paisley, UK), 0.5 ng reverse transcription primer for 60 min at 37°C.
  • the RT primer was composed of an anchored oligo (dT) primer with a specific 5' heel sequence: CTCTCAAGGATCTTACCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (A,G,C) .
  • Second-strand cDNA synthesis was initiated by incubation of the first-strand cDNA with 1 ng of a primer consisting of (5' to 3'); a 20 base sequence selected due to its absence from the mammalian databases, a stretch of five random nucleotides and a defined pentameric sequence (CTGCATCTATCTAATGCTCCNNNNNCGAGA where N represents C, G, T or A) for 15 mins at 50°C under amplification conditions described below.
  • a primer consisting of (5' to 3'); a 20 base sequence selected due to its absence from the mammalian databases, a stretch of five random nucleotides and a defined pentameric sequence (CTGCATCTATCTAATGCTCCNNNNNCGAGA where N represents C, G, T or A) for 15 mins at 50°C under amplification conditions described below.
  • the subsequent PCR between the heel sequence of the oligo (dT) primer and the arbitrary primer closest to the 5' end ensures amplification of cDNA sequences complementary to the 3' ends of the polyA tail.
  • primer extension was performed at 72°C for 10 min using AmpliTaq DNA polymerase (0.35 units, Applied Biosystems, Warrington, UK) in PCR buffer containing 67 mM Tris HC1 (pH 8.3), 4.5 mM MgCl 2 and 0.5 mM dNTPs. Subsequently, 0.4 ng of 3' heel primer
  • Riboprotein 1.5 RPL5 U14966 GAACAGCGTAACTCCAGACATG CCGCTCCTGAGCTCTGAG
  • Riboprotein L21 RPL21 U14967 GTGCGTATTGAGCACATAAAGC TTCCAGCAGCTCAGGCTC a RPL27a U14968 GACAAATTGTGGACTTTGGTCA ATGTGCTTCAAGCCACCAG
  • HSKPQZ7 M81806 GGAACTTCCTCTGGGAACCT GGAGGTCAAGTCAAGCTCCA 3-
  • G-3-PDH M33197 CGACCACTTTGTCAAGCTCA rogenese AGGGGTCTACATGGCAACTG a-actin ACTB AB004047 CGTGGACATCCGTAAAGACC ACATCTGCTGGAAGGTGGAC
  • CD19 CD19 X13312 CCAACCTCTGGAGCAATGTT GGAATACAAAGGGGACTGGA
  • CD79A CD79A L32754 CTTCTGGGGGCTTCCTTAGT GTTAGGAGGTGGGGCAGTTT3
  • CD2 CD2 M16445 TCTTCGAACTCAGCCATGTG GGCTGCTTGTAGTGAGACCC
  • Beta-actin Aclb V01217 CATCGATGCCCTGAGTCC ACACCTCAAACCACTCCCAG
  • Alpha-tubulin Tuba V01226 CAGTGGTACGTGGGTGAGG TTTGACATGATACAGGGACTGC
  • Neurokinin receptor 1 Nkl J05097 CTGGAAAGAGGAGCCTTGTG CTGAGACGGAAAGGAACAGC c GO Neurokinin receptor 2 Nk2 M31838 TTCTGCAGTGAGGAGCTGG TTGGCTTTCAGAGGGCAC co
  • Adora2a M91214 TCTGACCAACAAAGCTGGC TGGAAGGAAAGGCAGTAGTCA m O m .a:
  • sucrase- isomaltase and CD2 were included as negative controls as they were not expected to be expressed in these cells since their mRNAs have only previously been observed in the gastrointestinal tract (Chadrasena G et al (1992), Cell Mol Biol, 38:243- 254) and in populations of T-cells (Sewell W et al (1986) Proc Natl Acad Sci USA, 83:8718-8722), respectively.
  • Another set of primers, designed to amplify an intronic sequence from a gene found in the Xq 2.5 region were used to detect genomic contamination.
  • Figure 3 clearly demonstrates the reproducibility of the gene-specific assays following amplification from single cells, with each of 11 housekeeping gene assays being detectable in duplicate reactions on each of four lymphoblastoma cells.
  • Such variability in gene expression has been encountered in other cell groups thought to be homogeneous (O'Dowd, D K & Smith M A (1996), Mol Neurobiol, 13:199-211).
  • the power of this technique lies in its potential to facilitate expression profiling of cells derived from complex cell populations, even when they form only a small proportion of the population as a whole, and in its ability to detect low abundance transcripts.
  • NK neurokinin
  • adenosine A 2a receptor were investigated in single striatal cholinergic interneurons which constitute a small fraction of the total cellular mass of the striatum.
  • the NK1 receptor is widely accepted as being expressed in these cells (Kawaguchi Y et al (1995), Trends in Neurosci, 1_8: 527-535) .
  • the expression of this gene was examined as an example of an mRNA species expressed at far lower levels than housekeeping genes.
  • NK1 receptor mRNA was detected in all the cholinergic neurons tested, although the NK2 and NK3 receptors were not, confirming that Substance P exerts its effects on these cells via the NK1 receptor (Bell M I et al (1998), Neurosci in press).
  • the cytoplasm from large cells was gently aspirated under visual control into- a patch-clamp recording electrode until at least 40% of the somatic cytoplasm had been collected.
  • the electrode was then withdrawn from the cell to form an outside-out patch which prevented contamination when the electrode were forced into a microtube and reverse transcribed, subjected to 3' cDNA amplification, and 2.5% of the product used in each gene specific PCR reaction.
  • TPEA-PCR permits expression profiling of single cells
  • the analysis of a complex cell system can be made in vivo .
  • Striatal cholinergic interneurons are readily identifiable in rat brain slices due to their large size (>30 ⁇ m diameter) when compared to surrounding cell types, which are predominately medium spiny neurons ( ⁇ 15 ⁇ m diameter) .
  • electrophysical characterisation of the cells Lee K et al (1997) J Neurochem, 69:1774- 1776)
  • cytoplasmic samples were taken using a patch pipette, reverse transcribed and the cDNA amplified. The expression of a variety of genes of was then investigated (two representative expression profiles of cholinergic interneurons are shown in Figure 4).
  • NK neurokinin

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Abstract

L'invention concerne un procédé permettant -'établir le profil d'expression de cellules isolées. Le procédé utilise une première amorce à « talon » (heeled) pour la transcription inverse d'ARNm dans un échantillon afin de produire des espèces d'ADNc à premier brin, et puis une deuxième population d'amorces à « talon » pour générer des ADNc à second brin. La partie « non-talon » (non-heeled) des secondes amorces « à talon » peuvent s'hybrider aux premiers brins transcrits inverses d'espèces d'ADNc, à raison d'au moins un brin sur toutes les longueurs correspondantes. La présence de séquences aléatoires et de séquences présélectionnées dans les secondes amorces permet de créer, à partir d'ARNm cellulaires, un profil d'ADNc qualitativement plus uniforme et donc plus représentatif. --
PCT/GB1999/002579 1998-08-05 1999-08-05 Transcription inverse, amplification, et leurs amorces WO2000008208A2 (fr)

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EP1275738A1 (fr) * 2001-07-11 2003-01-15 Roche Diagnostics GmbH Procédé pour la synthèse aléatoire et l'amplification d'ADNc
EP1348762A1 (fr) * 2000-12-12 2003-10-01 Aisin Seiki Kabushiki Kaisha Methode d'analyse d'expression genique
WO2004007766A1 (fr) * 2002-07-10 2004-01-22 Cambridge Biotechnology Ltd. Methode d'amplification d'acide nucleique
US6692918B2 (en) * 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
EP1436404A2 (fr) * 2001-09-19 2004-07-14 Alexion Pharmaceuticals, Inc. Matrices transgeniques et leurs utilisations dans l'amplification d'amorce unique
US6946251B2 (en) 2001-03-09 2005-09-20 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences using RNA-DNA composite primers
US7176025B2 (en) 2002-03-11 2007-02-13 Nugen Technologies, Inc. Methods for generating double stranded DNA comprising a 3′ single stranded portion and uses of these complexes for recombination
US7351557B2 (en) 2001-03-09 2008-04-01 Nugen Technologies, Inc. Amplification of RNA sequences using composite RNA-DNA primers and strand displacement
US7414111B2 (en) 2001-09-19 2008-08-19 Alexion Pharmaceuticals, Inc. Engineered templates and their use in single primer amplification
US7771934B2 (en) 2000-12-13 2010-08-10 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US8143001B2 (en) 2003-12-29 2012-03-27 Nugen Technologies, Inc. Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids
US8465950B2 (en) 2003-04-14 2013-06-18 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US9206418B2 (en) 2011-10-19 2015-12-08 Nugen Technologies, Inc. Compositions and methods for directional nucleic acid amplification and sequencing
US9650628B2 (en) 2012-01-26 2017-05-16 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library regeneration
US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
US9822408B2 (en) 2013-03-15 2017-11-21 Nugen Technologies, Inc. Sequential sequencing
US9957549B2 (en) 2012-06-18 2018-05-01 Nugen Technologies, Inc. Compositions and methods for negative selection of non-desired nucleic acid sequences
US10102337B2 (en) 2014-08-06 2018-10-16 Nugen Technologies, Inc. Digital measurements from targeted sequencing
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US11028430B2 (en) 2012-07-09 2021-06-08 Nugen Technologies, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system

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WO2001006004A3 (fr) * 1999-07-19 2001-08-09 Univ Cambridge Tech Procede d'amplification de sequences d'acide nucleique peu abondantes et moyens mis en oeuvre pour ce procede
WO2001006004A2 (fr) * 1999-07-19 2001-01-25 Cambridge University Technical Services Ltd. Procede d'amplification de sequences d'acide nucleique peu abondantes et moyens mis en oeuvre pour ce procede
US6692918B2 (en) * 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
EP1348762A1 (fr) * 2000-12-12 2003-10-01 Aisin Seiki Kabushiki Kaisha Methode d'analyse d'expression genique
EP1348762A4 (fr) * 2000-12-12 2005-08-10 Masumi Abe Methode d'analyse d'expression genique
US8334116B2 (en) 2000-12-13 2012-12-18 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US7771934B2 (en) 2000-12-13 2010-08-10 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US8071311B2 (en) 2001-03-09 2011-12-06 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US9181582B2 (en) 2001-03-09 2015-11-10 Nugen Technologies, Inc. Compositions for amplification of RNA sequences using composite primers
US6946251B2 (en) 2001-03-09 2005-09-20 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences using RNA-DNA composite primers
US7354717B2 (en) 2001-03-09 2008-04-08 Nugen Technologies, Inc. Methods and kits for amplification of RNA sequences using composite primers
US7351557B2 (en) 2001-03-09 2008-04-01 Nugen Technologies, Inc. Amplification of RNA sequences using composite RNA-DNA primers and strand displacement
EP1275738A1 (fr) * 2001-07-11 2003-01-15 Roche Diagnostics GmbH Procédé pour la synthèse aléatoire et l'amplification d'ADNc
EP1436404A4 (fr) * 2001-09-19 2006-07-05 Alexion Pharma Inc Matrices transgeniques et leurs utilisations dans l'amplification d'amorce unique
US7414111B2 (en) 2001-09-19 2008-08-19 Alexion Pharmaceuticals, Inc. Engineered templates and their use in single primer amplification
US7306906B2 (en) 2001-09-19 2007-12-11 Alexion Pharmaceuticals, Inc. Engineered templates and their use in single primer amplification
EP1436404A2 (fr) * 2001-09-19 2004-07-14 Alexion Pharmaceuticals, Inc. Matrices transgeniques et leurs utilisations dans l'amplification d'amorce unique
US7176025B2 (en) 2002-03-11 2007-02-13 Nugen Technologies, Inc. Methods for generating double stranded DNA comprising a 3′ single stranded portion and uses of these complexes for recombination
AU2003254445B2 (en) * 2002-07-10 2006-06-15 Cambridge Biotechnology Ltd. Nucleic acid amplification method
WO2004007766A1 (fr) * 2002-07-10 2004-01-22 Cambridge Biotechnology Ltd. Methode d'amplification d'acide nucleique
US9175325B2 (en) 2003-04-14 2015-11-03 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US8465950B2 (en) 2003-04-14 2013-06-18 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US8143001B2 (en) 2003-12-29 2012-03-27 Nugen Technologies, Inc. Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids
US8852867B2 (en) 2005-09-07 2014-10-07 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
US9206418B2 (en) 2011-10-19 2015-12-08 Nugen Technologies, Inc. Compositions and methods for directional nucleic acid amplification and sequencing
US10036012B2 (en) 2012-01-26 2018-07-31 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
US9650628B2 (en) 2012-01-26 2017-05-16 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library regeneration
US10876108B2 (en) 2012-01-26 2020-12-29 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
US9957549B2 (en) 2012-06-18 2018-05-01 Nugen Technologies, Inc. Compositions and methods for negative selection of non-desired nucleic acid sequences
US11028430B2 (en) 2012-07-09 2021-06-08 Nugen Technologies, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US11697843B2 (en) 2012-07-09 2023-07-11 Tecan Genomics, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US10619206B2 (en) 2013-03-15 2020-04-14 Tecan Genomics Sequential sequencing
US10760123B2 (en) 2013-03-15 2020-09-01 Nugen Technologies, Inc. Sequential sequencing
US9822408B2 (en) 2013-03-15 2017-11-21 Nugen Technologies, Inc. Sequential sequencing
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US11098357B2 (en) 2013-11-13 2021-08-24 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US11725241B2 (en) 2013-11-13 2023-08-15 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
US10102337B2 (en) 2014-08-06 2018-10-16 Nugen Technologies, Inc. Digital measurements from targeted sequencing
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system

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