WO2008150937A2 - Real-time pcr - Google Patents

Abstract

The disclosure provides for the use of real-time PCR to detect the expression level(s) of genetic material in a sample. The expression level of any gene sequence, in the form of messenger RNA (mRNA) molecules, may be detected and/or quantified. The disclosed methods may be applied to RNA profiling, or determining the expression levels of multiple gene sequences in a biological sample, including a sample containing relatively degraded RNA, such as an FFPE sample.

Description

REAL-TIME PCR

RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional Patent Application 60/940,686, filed May 29, 2007, which is hereby incorporated by reference in its entirety as if fully set forth.

FIELD OF THE DISCLOSURE

The disclosure relates to real-time PCR to detect the expression level(s) of genetic material in a sample. In particular, the expression level of any gene sequence, in the form of messenger RNA (mRNA) molecules, may be detected and/or quantified. The disclosed methods may be used for RNA profiling, or determining the expression levels of multiple gene sequences in a biological sample, including a sample containing relatively degraded RNA.

BRIEF SUMMARY OF THE DISCLOSURE

The disclosure includes methods to use real-time polymerase chain reaction (PCR) to qualitatively detect, or quantitatively determine, the expression of one or more gene sequences in an RNA containing biological sample. The methods include 1) combined cDNA synthesis, annealing of oligonucleotides followed by their extension, and ligation of the extension product; 2) use of PCR with primer sequences present in the oligonucleotides; and 3) real-time PCR mediated assaying of an RNA sequence present within the cDNA molecule.

The disclosed methods may be used to assay for the expression of RNA sequences in a variety of cell containing biological samples, such as archival, freshly frozen, and fresh samples. In most cases, the methods are suitable for use as a robust clinical assay to detect expression level(s) for comparison to archival or other referential samples. The robustness of the methods includes the feature of consistency between samples and assay runs, which are provided in part by the real-time PCR portions of the method. With the use of real-time PCR to detect the product PCR amplicon, the methods do not rely upon a hybridization reaction to the amplicon as the means to determine the relative amount of a RNA sequence of interest. A hybridization reaction directed to a target or unique sequence in the amplified material is necessarily limited by the technical challenges of limited linearity (of about 2 to about 3 logs) and variability between assay runs. Both of these limitations are reduced and/or eliminated by the disclosed methods, where real-time PCR has been highly regarded as a standard for quantifying mRNA expression because of its linearity (about 8 to 9 logs), sensitivity to a single copy, and reproducibility between independent runs or experiments.

The disclosure includes the identification and use of gene expression patterns (or profiles or "signatures") by use of the disclosed method. The gene expression profiles may be viewed as being embodied in a nucleic acid expression, or mRNA, format which may be elucidated, interrogated, or studied by use of the disclosed method.

In a first aspect, the disclosure includes a method to identify, and then detect, gene expression profiles by use of real-time PCR. The method may be used to detect the presence, and optionally the amount of, an RNA sequence present as a part of an RNA molecule or a counterpart cDNA molecule. In many embodiments, the method includes synthesizing a first, single-stranded cDNA molecule as a counterpart to an RNA molecule containing an RNA sequence to be detected. The synthesis can be readily accomplished by reverse transcription of the RNA molecule by methodologies known to the skilled person, such as the use of a primer that hybridizes to the polyadenylated (polyA) tail of an RNA molecule or the use of a primer that hybridizes to a particular sequence within the RNA molecule via basepair complementarity. In the case of a polyA RNA molecule, the primer may contain an oligo dT sequence to permit hybridization to the polyA sequence.

Of course the RNA molecule may be one of a plurality of RNA molecules present in a sample. Non-limiting examples include nucleic acid containing samples from a plant, animal, bacterium, or virus. In some embodiments, the sample is obtained from one or more cells from an animal or a human patient.

In most cases, the method further includes annealing first and second DNA oligonucleotides to the single-stranded cDNA molecule. The cDNA molecule, which is complementary to all or part of the original RNA molecule template and is complementary to the RNA sequence to be detected, serves as a template upon which the first oligonucleotide may be extended. The first oligonucleotide contains a sequence complementary to the cDNA molecule and contains a forward primer (FP) sequence at its 5' end. The sequence complementary to the cDNA molecule may be complementary to all or part of the cDNA molecule's sequence that is complementary to an RNA sequence to be detected present in the original RNA molecule. The sequence complementary to the cDNA molecule would thus include all or part of an RNA sequence to be detected in the form of a DNA counterpart to the RNA sequence. So if the RNA sequence to be detected included the sequence AUG, the sequence complementary to the cDNA molecule would include the sequence ATG.

Alternatively, the sequence complementary to the cDNA molecule may not contain any portion of the RNA sequence to be detected in the form of a DNA counterpart to the RNA sequence. But in such a case, the sequence complementary to the cDNA molecule may be extended from the 3' end of the first oligonucleotide (such as by a DNA polymerase activity under suitable conditions) to include a portion containing all or part of an RNA sequence to be detected in the form of a DNA counterpart to the RNA sequence.

And while the FP sequence is optionally also complementary to the cDNA molecule, the FP sequence is not necessarily complementary to the cDNA molecule and may contain a unique sequence to facilitate subsequent hybridization to a PCR primer sequence and amplification by PCR.

The second oligonucleotide contains, at its 5' end, a sequence complementary to the cDNA molecule. The second oligonucleotide also contains, at its 3' end, a sequence that is not complementary to the cDNA molecule such that extension, via use of the cDNA molecule as a template, is not generally possible. This sequence at the 3' end may be used as a reverse primer (RP) sequence as described herein. In the second oligonucleotide, between the 5' sequence complementary to the cDNA molecule and the RP sequence at the 3' end, there is optionally a detectable reporter (or "address") sequence which can be subsequently detected as described herein.

The first and second oligonucleotides anneal to non-overlapping portions of the cDNA molecule, and in an orientation such that extension from the 3' end of the first oligonucleotide (such as by a DNA polymerase activity under suitable conditions) lengthens the first oligonucleotide toward the 5' end of the second oligonucleotide. In some embodiments, the first and second oligonucleotides are non-overlapping but spaced so that the 3' end of the first oligonucleotide may be ligated to the 5' end of the second oligonucleotide without the need for strand extension of the first oligonucleotide.

In embodiments requiring the extension of the first oligonucleotide, the method includes said extension before ligation with the second oligonucleotide. In embodiments where no extension is necessary, the ligating of the two oligonucleotides may be performed by use of a ligase activity under suitable conditions. Ligation of the two oligonucleotides forms a second DNA strand with a portion complementary to the cDNA molecule. That portion will include a DNA counterpart sequence to all or part of an RNA sequence to be detected. The second DNA strand will serve as a DNA template for subsequent manipulation and analysis.

More particularly, the second DNA strand will include the FP sequence from the 5' end of the first oligonucleotide and the RP sequence from the 3' end of the second oligonucleotide. These primer sequences are then used to amplify the second DNA strand. The amplification may be by PCR to produce multiple copies of an amplicon, followed by real-time PCR as described below, or may be by real-time PCR alone.

In cases including use of PCR, the PCR conditions may include the use of forward and reverse primers that are exactly complementary to the FP and RP sequences present in the second DNA strand, respectively. Alternatively, the forward and/or reverse primers may be complementary to portions of the FP and/or RP sequences sufficient to permit PCR.

The amplicon will include a DNA counterpart sequence to all or part of an RNA sequence to be detected. Optionally, the amplicon will include a detectable reporter (or "address") sequence present from the second oligonucleotide. Following PCR, the RNA sequence to be detected, as present in the amplicon, can then be detected by use of real-time PCR that detects either, or both, strands of the amplicon. In some embodiments, the detection by realtime PCR may be mediated by detecting a reporter (or "address") sequence present in the amplicon. In many embodiments, the real-time PCR may include the use of a probe that hybridizes to a sequence within the region amplified by the primers used. In some embodiments, the probe is complementary to (or overlaps with) a portion of one of the two primers used. A probe may be labeled with a donor fluorescent moiety and a second quencher or acceptor fluorescent moiety. As a non-limiting example, the probe may be a Taqman probe. In cases where the second DNA strand is amplified by real-time PCR without first being amplified by PCR, the real-time PCR conditions may include the use of forward and reverse primers as described herein for standard PCR. Additionally, the real-time PCR will include the use of probe as described herein.

In alternative embodiments, the method may include the labeling of the cDNA molecule complementary to all or part of the RNA molecule to be detected. Non-limiting examples include biotinylation of the cDNA molecule, such as by use of a biotinylated primer during reverse transcription.

In additional embodiments, the cDNA molecule is isolated or immobilized prior to use. Non-limiting examples include isolation, or immobilization on a solid support, via a biotin label present on the cDNA molecule. Methods to isolate or immobilize labeled cDNA molecules are known to the skilled persona and include the use of strepavidin linked beads or columns where the label is biotin. The isolation or immobilization may advantageously occur prior to the annealing of any oligonucleotide to the cDNA molecule.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic illustration of one embodiment of the disclosure.

DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSURE

Definitions of terms as used herein:

A gene expression "pattern" or "profile" or "signature" refers to the relative expression of a gene between two or more cells or cell types, such as that between normal and diseased or cancerous cells.

A "gene" is a polynucleotide that encodes a discrete product, whether RNA or proteinaceous in nature. It is appreciated that more than one polynucleotide may be capable of encoding a discrete product. The term includes alleles and polymorphisms of a gene that encodes the same product, or a functionally associated (including gain, loss, or modulation of function) analog thereof, based upon chromosomal location and ability to recombine during normal mitosis.

A "polynucleotide" or "oligonucleotide" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications including labels known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the polynucleotide.

A "probe" is a suitably labeled polynucleotide or oligonucleotide for use in real-time PCR. A non-limiting example is a Taqman probe.

The term "amplify" is used in the broad sense to mean creating an amplification product can be made enzymatically with DNA or RNA polymerases. "Amplification," as used herein, generally refers to the process of producing multiple copies of a desired sequence, particularly those of a sample. "Multiple copies" mean at least 2 copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence.

Because the disclosed methods rely upon the identification of gene sequences that are expressed, a polynucleotide containing a sequence that is unique is used as a primer or probe disclosed herein. Exemplary polynucleotides of this type contain at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, or at least about 32 consecutive basepairs of a sequence that is not found in other polynucleotides present with the sequence complementary to the primer or probe. The term "about" as used in the previous sentence refers to an increase or decrease of 1 from the stated numerical value. Additional embodiments include polynucleotides of at least or about 50, at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, or at least or about 400 basepairs of a gene sequence that is not found in other polynucleotides present with the sequence complementary to the primer or probe. The term "about" as used in the preceding sentence refers to an increase or decrease of 10% from the stated numerical value.

Such polynucleotides may also be referred to as polynucleotide probes that are capable of hybridizing to sequences of a gene, or unique portion(s) thereof, to be detected as described herein. The sequences may be those of an mRNA molecule encoded by a gene, the corresponding cDNA to such an mRNA molecule, and/or amplified versions of such sequences. Alternatively, the polynucleotide may be an oligonucleotide or primer as described herein.

The term "support" refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass slides.

As used herein, a "biological sample" or "cell containing sample" refers to a sample of cells or tissue isolated from an individual. The sample may be from material removed via a surgical or non-surgical procedure, including, but not limited to, surgical biopsy. Such samples are primary isolates (in contrast to cultured cells) and may be collected by any suitable means recognized in the art.

"Expression" and "gene expression" include transcription and/or translation of nucleic acid material.

As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as hybridization, strand extension, and the like, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions also depend on what event is desired, such as hybridization, cleavage, strand extension or transcription.

Sequence "mutation," as used herein, refers to any sequence alteration in the sequence of a gene disclosed herein interest in comparison to a reference sequence. A sequence mutation includes single nucleotide changes, or alterations of more than one nucleotide in a sequence, due to mechanisms such as substitution, deletion or insertion. Single nucleotide polymorphism (SNP) is also a sequence mutation as used herein. Because the present invention is based on the relative level of gene expression, mutations in non-coding regions of genes as disclosed herein may also be assayed in the practice of the invention.

"Detection" includes any means of detecting, including direct and indirect detection of gene expression and changes therein. For example, "detectably less" products may be observed directly or indirectly, and the term indicates any reduction (including the absence of detectable signal). Similarly, "detectably more" product means any increase, whether observed directly or indirectly.

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

Embodiments of the disclosure

With reference to Figure 1, select embodiments of the disclosure are provided. In Figure 1, total RNA may be used as a starting material. In alternative embodiments, the starting material may be mRNA, polyadenylated RNA, or RNA that has been purified or isolated in whole or in part. The RNA may be in a sample, or aliquot thereof, from a cell or cell containing material, such as a biological sample from a human patient or animal subject.

The RNA containing material is then used as a template for cDNA synthesis of a first, complementary, cDNA molecule (single stranded). Where the RNA is total RNA, the cDNA will be representative of the reverse transcribed portion of the RNA population. In some embodiments, the RNA contains polyadenylated RNA, and the reverse transcription comprises use of an oligo dT primer and optionally random primers that are complementary to a multiplicity of RNA sequences and so molecules. In some embodiments, the random primers are biotinylated or otherwise labeled in an analogous manner that allows for subsequent purification and/or capture of the cDNA molecules. In other embodiments, it is the oligo dT, rather than random, primer that is biotinylated or otherwise labeled for purification and/or capture.

The cDNA molecule(s) are then used as a template, or starting material, for the annealing of DNA oligonucleotides comprising sequences complementary to sequence(s of interest (query sequence or sequences) as present on an RNA molecule that has been reverse transcribed into the cDNA. As schematically illustrated in Figure 1, at least two oligonucleotides are used. A first oligonucleotide comprises a "Pl" primer (or forward primer) sequence at its 5' end to facilitate PCR amplification as discussed below. This first oligonucleotide is extended to allow ligation to the second oligonucleotide, which comprises a "P2" primer (or reverse primer) sequence at its 3' end to facilitate PCR amplification as discussed below. The "P2" containing oligonucleotide optionally includes, as shown in Figure 1, an "address" sequence which permits an alternative means of detection by real-time PCR as discussed below.

The "Pl" and "P2" containing oligonucleotide pairs may be used optionally in combination with one or more additional pairs comprising sequences complementary to one or more other sequences of interest present in other RNA molecules or other regions of the same RNA molecule. Stated differently, the disclosure includes the use of additional pairs of oligonucleotides to permit multiplex detection of more than one RNA sequence to be detected. In some embodiments, the acts of annealing, extending, and ligating may be performed in a single tube or container for the reaction.

As indicated by the lower portion of Figure 1, the resultant, ligated molecules are amplified by PCR with the "Pl" and "P2" primers (or common primers) to produce copies of an amplicon for subsequent real-time PCR analysis. Where more than one RNA sequence is queried by the more than one oligonucleotide pairs used, the PCR amplification will co-amplify all of the RNA sequences that are queried. This co-amplification reaction may advantageously be conducted in a single tube or container as well.

The detection of each RNA sequence in the amplified material (amplicons) may be performed in single-plex (where only one RNA sequence of interest is assayed) or in multi-plex (where more than one RNA sequence of interest is assayed) format using the common primers with an internal real-time PCR probe that is either within the targeted RNA sequence (not shown in Figure 1) or a probe that detects the "address" sequence as shown in Figure 1. In either embodiment, the probe may be complementary to the targeted RNA or "address" sequence or the same as the targeted RNA or "address" sequence as a design choice. The complementarity between the strands of the amplicons allows either strand to be detected in most cases. In further embodiments, a disclosed method may be used with oligonucleotides, for annealing, extension, ligation, and subsequent PCR amplification, that have already been designed or selected for use with other protocols. Non-limiting examples include those where the amplicon(s) are detected by hybridization to a probe, optionally immobilized on a solid support.

A disclosed method may be advantageously used with samples containing degraded or compromised RNA as the input, or starting material. Non-limiting examples include cell containing samples that have been formalin fixed and paraffin-embedded (FFPE samples). Other examples of starting materials include frozen or fresh cell or tissue samples.

A disclosed method may be considered to provide a means to analyzing global, or near global, gene expression from cells. In some cases, a method is used with a single cell or a homogenous cell population, such as one dissected away from, or otherwise isolated or purified from, contaminating cells beyond that possible by a simple biopsy. In other embodiments, a method is used to analyze expression of polyadenylated RNA from a human patient or animal subject.

A method of the disclosure may be used with the removal of contaminating cells (such as infiltrating lymphocytes or other immune system cells) that do not provide relevant or desirable information regarding the expression level(s) of interest. The contaminating cells are not present to possibly affect the genes identified or the subsequent analysis of gene expression. Such contamination is present where a biopsy containing many cell types is used to generate gene expression profiles.

Preferred animals for the application of the present invention are mammals, particularly those important to agricultural applications (such as, but not limited to, cattle, sheep, horses, and other "farm animals"), animal models of prostate cancer, and animals for human companionship (such as, but not limited to, dogs and cats).

In additional embodiments, a disclosed method may be used to profile mRNAs that are highly degraded because the primers (P1/P2) and probe may be selected to target a relatively short mRNA sequence. In some cases, the sequence may be about 40, about 42, about 44, about 46, about 48, about 50 or about 52 or longer bases in length. In other embodiments, the sequence may be about 59, about 61, about 63, about 65, about 67, about 69 or longer in length.

Where a probe detects a sequence present in the RNA, the sequence may that present near a polyadenylated tail, or 3' end, of the RNA molecule. In some embodiments, this may be a sequence that is in whole, or in part, within the 3' untranslated region of the RNA molecule. In other embodiments, the sequence may include both translated and untranslated sequences.

Alternatively, a probe that detects an "address" sequence may be used as described herein. The "address" sequence may be any suitably unique sequence selected by a skilled practitioner to facilitate its detection. The presence of the "address" sequence adjacent to a targeted RNA sequence in the amplicon allows detection of the "address" sequence to serve as an indirect detection of the targeted sequence.

Real-time PCR

Real-time PCR allows quantitative measurements of a nucleic acid molecule, such as a DNA molecule, to be made with much more precision and reproducibility because it relies on threshold cycle (CT) values determined during the exponential phase of PCR rather than endpoint measurements.

One type of real time PCR, using a primer pair and a fluorogenic (dark-hole-quencher) probe (such as 5'-6-FAM/MGB), is based on the hydrolysis of the fluorogenic probe. The probe contains a 5'-fluorophore and a 3'-quencher and anneals to a specific target sequence between the upstream and the downstream primers in a PCR system. The 3'-terminus of the probe may be optionally blocked to prevent its use as a primer for strand extension. In the presence of the appropriate cycling conditions, the PCR reaction proceeds as the 5' to 3'-endonuclase activity of the thermal stable polymerase enzyme cleaves the fluorophore from the probe. Because the fluorophore is no longer in close proximity to the quencher its fluorescence becomes detectable. As the concentration of cleaved fluorophore in solution increases, the resultant fluorescent signal is monitored by real-time fluorometric analysis.

Fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The more templates present at the beginning of the reaction, the fewer number of cycles it takes to reach a point in which the fluorescence signal is first recorded as statistically significant above background, which is the definition of the (Ct) values.

So the disclosure includes the use of a real-time PCR probe that is labeled with a donor fluorescent moiety and a second quencher or acceptor fluorescent moiety. The detection methods of the real-time PCR assay further includes detecting the presence or generation of detectable fluorescence, and thus the absence or decrease in fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the quencher or acceptor fluorescent moiety in the PRRSV probe. The presence or generation of detectable fluorescence is usually indicative of the presence of the nucleic acid sequence being amplified, and the absence of detectable fluorescence is usually indicative of the absence of the sequence.

Detection of fluorescence is preferably the result of amplification using a (thermostable) polymerase enzyme having 5' to 3' exonuclease activity which cleaves the donor fluorescence moiety from the probe to result in a detectable signal. The locations of the donor and quencher or acceptor moieties on the probe may be such that FRET may occur between the two moieties. The disclosure contemplates the location of the donor moiety at or near the 5' end of the probe and the quencher or acceptor moiety at or near the 3' end of the probe with a separation of from about 14 to about 22 basepairs between the moieties, although other distances, such as from about 6, about 8, about 10, or about 12 basepairs may be used. Additional distances are about 14, about 16, about 18, about 20, or about 22 basepairs.

In alternative embodiments, a probe can include a nucleic acid sequence that permits secondary structure formation (such as a hairpin) that results in spatial proximity between the donor and the quencher or acceptor fluorescent moiety. Such a method does not require hydrolysis of the probe and has been referred to as the "molecular beacon" approach (see for example, Tyagi S et al. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14, 303-308).

In yet additional embodiments, a pair of probes is used, where one probe contains the donor moiety and the other probe contains the acceptor moiety. Such an assay still includes performing at least one cycling step of the real-time PCR, wherein a cycling step comprises amplification and hybridization. The hybridization includes contacting the target sequence with a pair of probes as described above. The method further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the two probes. The presence or absence of FRET is indicative of the presence or absence of a nucleic acid sequence in a sample. Such a method can further include determining the melting temperature between the amplification product and one or both of the probes. The melting temperature can confirm the presence or absence of a nucleic acid sequence.

In further embodiments, a nucleic acid binding dye is used in place of any nucleic acid probe. The dye-binding includes contacting the amplification product with a nucleic acid binding dye followed by detecting the presence or absence of binding of the nucleic acid binding dye to the amplification product. The presence of binding is usually indicative of the presence of the amplified nucleic acid sequence, and the absence of binding is usually indicative of the absence thereof. Non-limiting examples of nucleic acid binding dyes include SybrGreen LRTM., SybrGold.RTM., and ethidium bromide.

A representative donor fluorescent moiety is FAM or 6-FAM, and a representative quencher or acceptor fluorescent moiety is MGB. Other non-limiting examples of a donor moiety include fluorescein, HEX, TET, TAM, ROX, Cy3, Alexa, and Texas Red while non- limiting examples of a quencher or acceptor fluorescent moiety include TAMRA, BHQ (black hole quencher), LCTM. -RED 640 (LightCycler.TM.-Red 640-N-hydroxysuccinimide ester), and cyanine dyes such as CY5. As will be appreciated by a person skilled in the art, any pair of donor and quencher/acceptor moieties may be used as long as they are compatible such that transmission may occur from the donor to the quencher/acceptor. Moreover, pairs of suitable donors and quenchers/acceptors are known in the art and are provided herein. The selection of a pair may be made by any means known in the art and may be confirmed by routine and repetitive testing for energy transfer or quenching of fluorescence.

Having now fully described the inventive subject matter, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the disclosure and without undue experimentation. While this disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting the presence of an RNA sequence, said method comprising a) synthesizing a first, single stranded cDNA molecule by reverse transcription of at least one RNA molecule containing said RNA sequence; b) annealing a first and a second oligonucleotide to said cDNA molecule as a template, wherein said first oligonucleotide comprises a sequence complementary to said cDNA and a forward primer sequence at its 5' end; and said second oligonucleotide comprises, at its 5' end, a sequence complementary to said cDNA, which can be ligated to said first oligonucleotide upon its extension, and a reverse primer sequence at its 3' end; c) extending said first oligonucleotide to permit ligation with said second oligonucleotide; d) ligating said first and second oligonucleotides to form a DNA template comprising the forward and reverse primer sequences at its 5' and 3' ends, respectively; e) optionally amplifying said DNA template by PCR to produce amplicons; f) detecting said RNA sequence by real-time PCR of a sequence complementary to said cDNA, or the complement of said sequence.

2. The method of claim 1 wherein said at least one RNA molecule is obtained from a cell containing sample comprising a plurality of RNA molecules.

3. The method of claim 2 wherein said sample is a formalin fixed paraffin embedded (FFPE) sample of cells or tissue.

4. The method of claim 1 or 2 or 3 wherein said RNA molecule is polyadenylated and said reverse transcription comprises use of an oligo dT primer.

5. The method of claim 1 or 2 or 3 or 4 wherein said second oligonucleotide further comprises a detectable address sequence between the sequence complementary to said cDNA and said reverse primer sequence.

6. The method of claim 5 wherein said detecting is of said address sequence.

7. The method of claim 1 or 2 or 3 or 4 or 5 or 6 wherein said amplifying comprises PCR with a forward primer identical to said forward primer sequence.

8. The method of claim 1 or 2 or 3 or 4 or 5 or 6 wherein said amplifying comprises PCR with a reverse primer complementary to said reverse primer sequence.

9. The method of any preceding claim wherein said cDNA molecule is biotinylated, optionally by use of a biotinylated primer for reverse transcription.

10. The method of claim 9 wherein said biotinylated cDNA molecule is isolated prior to b).

11. The method of any preceding claim, wherein said detecting by real-time PCR comprises a Taqman probe.