miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

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.

Keywords: microRNA; primer design strategies; primer dimerization; primer specificity; qPCR efficiency; real-time PCR.

FIGURE 1.

Framework of miPrimer's design strategies. miPrimer includes two major systems: uni-system and specific-FR-system. The specific-FR-system contains three sequential design rules/functions, overlap, FPM (forward primer major), and RPM (reverse primer major), to reduce dimer issues and increase primer specificity. There are four design strategies highlighted in color, from left to right, uni-system, specific-FR-system^overlap, specific-FR-system^FPM, and specific-FR-system^RPM. A more detailed description of when and how to utilize the strategies is transcribed in the Materials and Methods section.

FIGURE 2.

qPCR efficiency of miRNA primers designed by miPrimer. (A) qPCR efficiency analysis of a serially diluted oligonucleotide hsa-miR-9-5p miRNA template against hsa-miR-9-5p-F primer and universal reverse primer designed by uni-system (four 10-fold dilutions; 2500 qPCR reactions per dilution). (B) qPCR efficiency analysis of a serially diluted synthetic oligonucleotide hsa-let-7b against hsa-let-7b-F/R designed by specific-FR-system^FPM (four 10-fold dilution; 2500 qPCR reactions per dilution). (C) qPCR efficiency analysis of a serially diluted artificial template using the universal reverse primer and a designed forward primer (four 10-fold dilutions; 2500 qPCR reactions per dilution). Each data point on the plot represents mean Cq ± SD from three replicates.

FIGURE 3.

Discrimination of miRNA family with primers designed by miPrimer. (A) Mature miRNA sequence of hsa-miR-18 family members. Sequence differences between members are indicated in red. Primers were designed by specific-FR-system^overlap. Discrimination of synthetic oligonucleotide hsa-miR-18 family members against homologous or heterologous miRNA primers was performed in 2500 qPCR reaction assays with Quark Biosciences’ DigiChip. Cq distributions of synthetic oligonucleotide hsa-miR-18 family members against homologous or heterologous miRNA were plotted based on the result of qPCR reactions. (B) Mature miRNA sequence of hsa-miR-16 family members. Sequence differences between members are indicated in red. Primers were designed by specific-FR-system^FPM. Discrimination of synthetic oligonucleotide hsa-miR-16 family members against homologous or heterologous miRNA primers was performed in 2500 qPCR reaction assay with Quark Biosciences’ DigiChip. Cq distributions of synthetic oligonucleotide hsa-miR-16 family members against homologous or heterologous miRNA were plotted based on the result of qPCR reactions. (C) Mature miRNA sequence of hsa-let-7 family members. Sequence differences between members are indicated in red. (D) No-template control (NTC) assay for each miRNA primer was performed to estimate the false positive rate.