{"doi":"10.1093/nar/gkab1153","title":"OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression","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.","journal":"Nucleic Acids Research","year":2021,"id":160651,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":34,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9553,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":265790,"name":"Na Ni","orcid":"0000-0003-1914-0604","position":1,"is_corresponding":false},{"id":227455,"name":"Hao Wang","orcid":"0000-0001-7475-0136","position":2,"is_corresponding":false},{"id":298744,"name":"Zongyue Zeng","orcid":"0000-0002-4333-5412","position":3,"is_corresponding":false},{"id":673928,"name":"Piao Zhao","orcid":"0000-0001-5543-6221","position":4,"is_corresponding":false},{"id":265788,"name":"Deyao Shi","orcid":"0000-0003-4131-1027","position":5,"is_corresponding":false},{"id":348078,"name":"Yinglin Xia","orcid":"0000-0001-6857-7437","position":6,"is_corresponding":false},{"id":340293,"name":"Connie Chen","orcid":"0000-0001-5016-6584","position":7,"is_corresponding":false},{"id":265780,"name":"Daniel A. Hu","orcid":"0000-0002-7979-3693","position":8,"is_corresponding":false},{"id":265779,"name":"Kevin Qin","orcid":"0000-0002-0424-4630","position":9,"is_corresponding":false},{"id":268972,"name":"William Wagstaff","orcid":null,"position":10,"is_corresponding":false},{"id":673929,"name":"David Qin","orcid":"0000-0001-9963-4641","position":11,"is_corresponding":false},{"id":673930,"name":"Bryce Hendren‐Santiago","orcid":"0000-0002-2558-7923","position":12,"is_corresponding":false},{"id":268973,"name":"Sherwin H. Ho","orcid":null,"position":13,"is_corresponding":false},{"id":674659,"name":"Rex C. Haydon","orcid":null,"position":14,"is_corresponding":false},{"id":673931,"name":"Hue H. Luu","orcid":"0000-0003-4562-0329","position":15,"is_corresponding":false},{"id":265793,"name":"Russell R. Reid","orcid":"0000-0002-1111-4500","position":16,"is_corresponding":false},{"id":337384,"name":"Le Shen","orcid":"0000-0002-4367-5476","position":17,"is_corresponding":false},{"id":673932,"name":"Hua Gan","orcid":"0000-0002-8261-684X","position":18,"is_corresponding":false},{"id":511586,"name":"Jiaming Fan","orcid":"0000-0001-8523-4188","position":19,"is_corresponding":false},{"id":265798,"name":"Tong‐Chuan He","orcid":"0000-0001-7721-3934","position":20,"is_corresponding":false},{"id":298747,"name":"Fang He","orcid":"0009-0009-8969-8348","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-18T23:44:48.240577Z","pmid":"34850128","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}