OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression. Issue 4 (25th November 2021)

• Record Type: Journal Article
• Authors: He, Fang; Ni, Na; Wang, Hao; Zeng, Zongyue; Zhao, Piao; Shi, Deyao; Xia, Yinglin; Chen, Connie; Hu, Daniel A; Qin, Kevin H; Wagstaff, William; Qin, David; Hendren-Santiago, Bryce; Ho, Sherwin H; Haydon, Rex C; Luu, Hue H; Reid, Russell R; Shen, Le; Gan, Hua; Fan, Jiaming; He, Tong-Chuan
• Is Part Of: Nucleic acids research. Volume 50:Issue 4(2022)
• Page Start: e22
• Page End: e22
• Publication Date: 2021-11-25
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkab1153
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: 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 products and quantified miRNAs with high efficiency. UHP-based RT-qPCR miRNA quantification was not affected by long transcripts. By analyzing 14 miRNAs, we demonstrated that UHP4 closely mimicked MsHPs in miRNA quantification. Fine-tuning experiments identified an optimized UHP (OUHP) mix with a molar composition of UHP2:UHP4:UHP6 = 8:1:1, which closely recapitulated MsHPs in miRNA quantification. Using synthetic LET7 isomiRs, we demonstrated that the OUHP-based qPCR system exhibited high specificity and sensitivity. Collectively, our results demonstrate that the OUHP system can serve as a reliable and cost-effective surrogate of MsHPs for RT-qPCR-based miRNA quantification for basic research and precision medicine.