MicroRNA (miRNAs) are 18-25 nucleotides highly conserved, non-coding RNAs involved in gene regulations. 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 sequences and miRNA family members which often only differ in sequences by one nucleotide.Here, we described 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 between 90% ~ 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating members of miRNA families as validated by qPCR assay 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.
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