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AssigneeNational University of Singapore
InventorsHeng Phon Too, Azlinda B. Anwar
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There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.
A method for detecting a target RNA molecule in a sample, the method comprising:
A method for detecting a target RNA molecule in a sample, the method comprising:
A method for detecting a target miRNA molecule in a sample, the method comprising:
Dependencies are inferred from the claim text.
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Nucleic acid amplification reactions: using modified primers or templates
Involving nucleic acids: nucleic acid amplification reactions
Nucleic acid amplification reactions: common amplification features
Oligonucleotides characterized by their use: mirna, sirna or ncrna
About 6 years 9 months of term left · ends 29 Jul 2033 (estimated)
The invention relates to methods of detecting a target RNA in a sample, including detecting a target miRNA in a sample.
MicroRNAs (miRNAs) are small non-coding RNAs (typically about 22 nucleotides in length) that were discovered as important post-transcriptional regulators of gene expression in metazoans (Bartel, 2004). While expression of miRNAs are critical in various physiological processes (Harfe, 2005; Miska, 2005; Carthew, 2006; Lindsay, 2008), dysregulation of miRNAs are implicated in pathologies of many human diseases such as cancer (Visone & Croce, 2009), muscle disorders (Chen et al., 2009a) and neurodegeneration (Hebert & De Strooper, 2009).
Recently, mature miRNAs were found to be remarkably stable in blood, and thus hold great promise as potential noninvasive biomarkers of human diseases (Chen et al., 2008; Mitchell et al., 2008). To date, hundreds of unique miRNAs have been identified in many species and each of these is predicted to regulate diverse target genes (Bartel, 2009). With the continual discovery of more miRNAs by both in silico prediction and in vivo validation (Mendes et al., 2009), profiling of miRNA expression remains an essential tool not only for assessment of distribution and regulation of miRNAs but also for identification of novel biomarkers and potential therapeutic targets.
The tables and figures, which illustrate, by way of example only, embodiments of the present invention, are as follows.
FIG. 1 is a schematic representation that provides an overview of the reverse transcription stage of the method of the invention.
FIG. 2 is a schematic representation that provides an overview of the amplification stage of the method of the invention.
There is presently provided a method for detection of RNA from a sample. The method may be used to detect any RNA species, and may be useful in detecting mature miRNA, including directly from a cell containing the miRNA.
The method is an RT-PCR (reverse transcription-PCR) amplification method that combines an RT oligonucleotide primer designed to have stem-loop secondary structure and a nucleotide modified to block DNA polymerase during an amplification reaction together with a pair hemi-nested amplification primers in the amplification reaction.
FIGS. 1 and 2 provide a schematic representation of an overview of the method; the 5′ and 3′ end of each nucleic acid molecule depicted is indicated. FIG. 1 represents a reverse transcription reaction and FIG. 2 represents an amplification reaction.