title: Universal stem-loop primer method for screening and quantification of microRNA.
abstract: RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called 'universal stem-loop primer' (USLP) with 8 random nucleotides instead of a specific sequence at the 3' end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 103 copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles.
authors.affiliation: Clinical Laboratory Diagnostic Centre, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Li-hong
authors.initials: LH
authors.lastname: Yang
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Si-lu
authors.initials: SL
authors.lastname: Wang
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Li-li
authors.initials: LL
authors.lastname: Tang
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Biao
authors.initials: B
authors.lastname: Liu
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Wen-le
authors.initials: WL
authors.lastname: Ye
authors.affiliation: Clinical Laboratory Diagnostic Centre, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Ling-ling
authors.initials: LL
authors.lastname: Wang
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Zhang-yang
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Meng-tao
authors.initials: MT
authors.lastname: Zhou
authors.affiliation: Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, Peoples Republic of China.
authors.forename: Bi-cheng
authors.initials: BC
authors.lastname: Chen
mesh_terms: D000328:Adult; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D005260:Female; D006801:Humans; D016867:Immunocompromised Host; D016030:Kidney Transplantation; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; C533097:MIR150, human; C524942:MIRN155 microRNA, human