LAUSR

MicroRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or gene silencing. Although quantitative real-time PCR (qRT-PCR) had been widely used for miRNAs quantification, a multiplex quantification method is demanding. In this study, we successfully detected 2 miRNAs (miR-505-3p and miR-21a-5p) and an internal control (miR-16-5p) with only one reaction based on competitive PCR (cPCR) with high sensitivity. For each miRNA, two stem-loop reverse transcription (RT) primers were designed to produce two different templates: the competitor cDNA and the target cDNA, which had similar sequences except for 3 nucleotides different in length. RNA from a control sample was reverse transcribed with the competitive RT primers of multiple genes. Samples for test were reverse transcribed with target RT primers to obtain target cDNAs. Target cDNA was mixed with competitor cDNA to be used as the template for a multiplex fluorescent cPCR reaction. The cPCR products were separated on polyacrylamide gel electrophoresis with ABI 377 DNA sequencer and each fluorescent peak was quantified by its intensity. In this method, we compared the expression level of miR-505-3p in two tissues (thalamus and tail) between C57BL/6J and C3H/HeJ mice. The results showed that in the thalamus, which had high abundance of miR-505-3p, both cPCR and SYBR Green based qRT-PCR provided a sensitive quantification outcome. However, in the tail, which had extremely low level of miR-505-3p, it could be steadily detected by cPCR even after 8 times dilution with a relatively high sensitivity, while qRT-PCR can’t detect any product only after 2 times dilution. The variation as low as 12.2% between samples could be clarified by cPCR, which could not be accomplished by qRT-PCR. This method enables multiplex, accurate and sensitive quantification of miRNAs with fewer precious RNA samples than qRT-PCR.