New micro ribonucleic acid quantitative PCR (polymerase chain reaction) detection method

Abstract

The new quantitative PCR detection process of micro RNAs (microRNAs) and small interference RNAs (siRNAs) includes: complementing the bases near the 5' end and the 3' end of retroprimer to form a stem-loop structure, combining the 3' end of retroprimer and target nucleic acid complementarily, and performing reverse transcription reaction with reverse transcriptase; amplifying cDNA obtained through the reverse transcription reaction with partial sequence of the retroprimer as one of the PCR primers; and real-time monitoring quantitative PCR with fluorescence labeled molecular beacon probe.

Description

Technical field [0001] The invention patent relates to the detection and expression quantitative analysis of microRNAs (microRNAs) and small interfering RNAs (siRNAs); the invention also relates to other human disease-related (such as cancer) ribonucleic acids such as non-coding RNA (non-coding RNA), mRNA Detection and expression quantification of splice variants (mRNA splice Variant). Background technique [0002] The present invention uses real-time quantitative PCR (Reai-time PCR) combined with molecular beacon probe (Molecular Beacon) technology, and is suitable for using RNA samples (such as total RNA, cell lysates, etc.) obtained by a variety of different preparation methods as templates, Specific detection and expression quantitative analysis of small interfering RNA, microRNA in biological cells and other ribonucleic acid target sequences. MicroRNA (microRNAs) [0003] For a long time, ribonucleic acid (RNA) was thought to only function to transcribe and translate ...

AI Technical Summary

Problems solved by technology

[0021] In 2003, Grad et al. reported the application of PCR technology to detect microRNAs. Since this method needs to recover microRNAs from the gel in advance, it is only suitable for miRNA cloning, and it is difficult to become a routine detection method in the laboratory (Grad et al.2003 , Mol. Cell 11:1253-1263)

In 2004, Schmittgen et al. reported a method for analyzing microRNAs precursor molecules by quantitative PCR, but this method still failed to solve the problem of detecting mature microRNAs (Schmittgen et al.2004, Nucleic Acids Res.32: e43 )

In 2005, Caifu Chen et al reported a method for quantitative PCR detection of microRNAs molecules using TaqMan-MGB probes. Compared with other methods, this method has the characteristics of high detection sensitivity and low sample consumption, but the TaqMan-MGB fluorescent probe The synthesis is expensive and its application is limited (Caifu Chen et al.2005, NucleicAcids Res.33: e179)

Images