Departments of Nephrology, Gastroenterology, Laboratory Diagnostic Medicine, and Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA.
Nucleic Acids Res. 2022 Feb 28;50(4):e22. doi: 10.1093/nar/gkab1153.
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.
微小 RNA（miRNA 或 miR）是一种单链、约 22 个核苷酸的非编码 RNA，可调节多种细胞过程。虽然有许多 miRNA 定量技术，但最近对 12 种商业平台的分析显示，在重复性、灵敏度、准确性、特异性和平台内/之间的一致性方面存在很大差异。在这里，我们开发了一种通用发夹引物（UHP）系统，该系统无需使用 miRNA 特异性发夹引物（MsHPs）进行基于定量逆转录 PCR（RT-qPCR）的 miRNA 定量。具体来说，我们分析了四个共享相同发夹结构但在 3'-末端锚定有两个、三个、四个和六个简并核苷酸的 UHPs（即 UHP2、UHP3、UHP4 和 UHP6），并发现这四个 UHPs 产生了稳健的 RT 产物，并以高效率定量 miRNA。UHP 基于 RT-qPCR 的 miRNA 定量不受长转录物的影响。通过分析 14 个 miRNA，我们证明 UHP4 在 miRNA 定量方面与 MsHPs 非常相似。微调实验确定了一个优化的 UHP（OUHP）混合物，其摩尔组成 UHP2：UHP4：UHP6 = 8：1：1，与 MsHPs 在 miRNA 定量方面非常相似。使用合成的 LET7 同型体，我们证明了 OUHP 基于 qPCR 的系统在 miRNA 定量方面具有高度特异性和灵敏度。总之，我们的研究结果表明，OUHP 系统可以作为 MsHPs 的可靠且具有成本效益的替代物，用于基础研究和精准医学中的基于 RT-qPCR 的 miRNA 定量。
