Abstract
MicroRNA (miRNA) detection by reverse transcription (RT) quantitative real-time PCR (RT-qPCR) is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3′-end length variants (“isomiRs”), little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3′-end miRNA isoforms can exhibit 3′-length specific regulatory functions, underlining the need to develop strategies to differentiate 3′-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20–21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3′-isomiRs.
Highlights
MicroRNAs are short RNAs controlling the translation of target messenger RNAs
It has previously been suggested that qPCR approaches relying on stem-loop or polyadenylation reverse transcription do not have the capacity to distinguish miRNA isoforms differing in their 3 -end (Wu et al, 2007; Schamberger and Orban, 2014; Magee et al, 2017)
Forward primers targeted to the shorter isoforms could all amplify the longer ones, which were anticipated since the longer isomiRs have perfect binding sites for these primers
Summary
MicroRNAs (miRNAs) are short RNAs controlling the translation of target messenger RNAs (mRNAs). They are processed from hairpin-like transcripts to their mature form through a sequential cleavage operated by Drosha in the nucleus, and Dicer, in the cytoplasm (Ha and Kim, 2014). Mature miRNA intracellular levels are under stringent control, as inefficient miRNA biogenesis and the resulting global decrease of miRNA levels are directly associated with the development of tumor cells (Melo et al, 2010; Wu et al, 2013). Intracellular miRNA levels are tightly controlled through the modulation of their expression and processing, with as many as 180 binding proteins interacting with select precursor miRNAs (pre-miRNAs) recently identified (Treiber et al, 2017)
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