Abstract
MicroRNAs (miRNAs) are 18–25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs’ short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences’ platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers.
Highlights
MiRNAs, short endogenous noncoding RNAs consisting of 18–25 nucleotides, are involved in post-transcriptional regulation by targeting the 3′ UTR of mRNA for mRNA degradation or translation inhibition (Lee et al 1993; Wightman et al 1993; Reinhart et al 2000; Bartel 2009)
The qPCR efficiency is impacted by a number of factors
To illustrate that the miRNA primers designed by miPrimer methodology (Fig. 1) can achieve excellent qPCR efficiency, a titration assay of four 10-fold serial dilutions was conducted with different miRNAs in various designing methods (Materials and Methods)
Summary
MiRNAs, short endogenous noncoding RNAs consisting of 18–25 nucleotides (nt), are involved in post-transcriptional regulation by targeting the 3′ UTR of mRNA for mRNA degradation or translation inhibition (Lee et al 1993; Wightman et al 1993; Reinhart et al 2000; Bartel 2009). MiRNAs are key regulators of gene expression networks, controlling diverse biological processes including proliferation, differentiation, apoptosis, metabolism, development, and host−pathogen interactions (Bueno et al 2008; Slaby et al 2009; Small and Olson 2011). MiRNAs have been demonstrated to be involved in cancer development (Calin and Croce 2006; Esquela-Kerscher and Slack 2006; Manikandan et al 2008; Zhang et al 2014; Ohtsuka et al 2015) and the progression of other diseases (Lu et al 2008; Jiang et al 2009; Li and Kowdley 2012; Mendell and Olson 2012).
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