biostudies-literatureHe FNational Center for Advancing Translational SciencesUniversity of Chicago Orthopaedics Alumni FundNCATS NIH HHSMabel Green Myers Research Endowment FundUniversity of Chicago Cancer CenterNCI NIH HHSNational Institutes of HealthNIGMS NIH HHSSHOCK Funde22https://www.ebi.ac.uk/biostudies/studies/S-EPMC8887422biostudies-literatureUnknown50(4)MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT prodNucleic acids researchOUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.PMC8887422UL1 TR000430P30CA014599DE030480CA226303T32 GM007281P30 CA014599Xia YHe FShen LLuu HHHo SHZeng ZHendren-Santiago BGan HWang HNi NChen CQin KHFan JZhao PHe TCWagstaff WHaydon RCShi DHu DAQin DReid RRfalseOUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT prod2022-01-01T00:00:00Z2022 Feb2025-04-26T04:39:34.251Z2025-04-06T11:12:45.015ZS-EPMC88874223485012810.1093/nar/gkab1153