A Design Method of Stem-loop Primer for Reverse Transcription of mir-505

Abstract

The invention relates to a design method of a stem-loop primer for miR-505 reverse transcription, which comprises the following the following steps: by using miRNA-505-3P gene as a specific transcript, designing several gradients corresponding to the quantity of stem base pairs of the reverse transcription stem-loop primers and the rules of the number of bases of the loop, and carrying out miRNA-505-3P reverse transcription and real time-PCR (polymerase chain reaction) detection to finally screen out the reverse transcription stem-loop primer with the optimal aging ration for the miRNA-505-3P gene specific reverse transcription. The method avoids the interference with the detection on the mature body, can clearly detect the respective influences of the structure of the loop bases and the structure of the stem bases on the reverse transcription efficiency of the reverse transcription stem-loop primer, and thus, can design the reasonable and efficient reverse transcription primer more easily.

Description

technical field [0001] The invention belongs to the field of miRNA detection, in particular to a method for designing a stem-loop primer for reverse transcription of miR-505. Background technique [0002] At present, the techniques for detecting miRNA mainly include Northern blot hybridization (Reinhart BJ, Weinstein EG, Rhoades MW, et al. , Weinstein EG, Rhoades MW, et al.MicroRNAs in plants[J].GenesDev,2002,16(13):1616^1626) and RT-PCR technology, but the most commonly used and most sensitive miRNA detection technology is qRT-PCR . The fluorescence signal concentration of the final product of qRT-PCR not only depends on the initial expression level of miRNA, but also is affected by reverse transcription and PCR amplification efficiency. In 2005, Chen et al. introduced the reverse transcription quantitative PCR technology into the detection of miRNA, and established a stem-loop reverse transcription quantitative PCR technology (stem-loop RT-PCR). He first designed a gener...

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Problems solved by technology

In 2010, Verduci L et al. found that miR-505 could regulate the proliferation and senescence / apoptosis of mouse embryonic fibroblasts by acting on its target ASF / SF2 (Verduci, L, Simili M, Rizzo M, Mercatanti A, Evangelista M et al. (2010) MicroRNA (miRNA)-mediated Interaction between Leukemia / Lymphoma-related Factor (LRF) and Alternative splicing factor / splicing factor 2 (ASF / SF2) affects mouse embryonic fibroblast senescence and apoptosis. Biol Chem, 285:39551-39563), Karni R et al. found that transfection of ASF / SF2 in many cells can activate part of the mTOR signaling pathway (Karni R, Hippo Y, Lowe SW, Krainer AR (2008) The splicing-factor oncoprotein SF2 / ASF activates mTORC1.ProcNatl Acad Sci USA 105(40):15323-15327), but the specific way ASF participates in the regulation of mTOR is unclear

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