title: Generating a long DNA fragment of the target ncRNA for quantitative polymerase chain reaction by combining ncRNA-oligos hybridization and oligos ligation.
abstract: The poor reproducibility of the reverse transcription combined with quantitative polymerase chain reaction (RT-qPCR) results in an unacceptable reliability of publications based on these data. We established a novel method, in which two short complementary DNA oligos were hybridized with target ncRNA molecules and linked by DNA ligase to obtain a long DNA strand (HL-DNA) replacing cDNA for qPCR detection (HL-qPCR). A series of diluted samples prepared from the same total RNA resource were measured by HL-qPCR and RT-qPCR respectively to acquire their relative concentration of RNU4-1, AK026510 and SNORA73B. For every tested sample, the relative concentration of RNU4-1, AK026510 and SNORA73B obtained by HL-qPCR instead of RT-qPCR is closer to its corresponding true value without significant difference, demonstrating that HL-qPCR exhibits higher accuracy compared with RT-qPCR. With three independent repeats, no significant difference was observed among AK026510/RNU4-1 values of four samples diluted from the same RNA resource, by employing HL-qPCR but not RT-qPCR. It strongly suggests that the good reproducibility of HL-qPCR results from the stable efficiency of HL-DNA production regardless of the concentration and individual features of ncRNA. The novel HL-qPCR could be applied for the regular relative ncRNA concentration detection in the future.
authors.affiliation: College of Biology, Hunan University, Changsha 410082, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: College of Biology, Hunan University, Changsha 410082, China. Electronic address: zhouzheng@hnu.edu.cn.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: College of Biology, Hunan University, Changsha 410082, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: College of Biology, Hunan University, Changsha 410082, China.
authors.forename: Zitian
authors.initials: Z
authors.lastname: Wang
authors.affiliation: College of Biology, Hunan University, Changsha 410082, China.
authors.forename: Huali
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Luo
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Run-Sheng
authors.initials: RS
authors.lastname: Chen
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D022661:RNA, Untranslated / Q000737:chemistry* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D048348:Reverse Transcription
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D022661:RNA, Untranslated; D012313:RNA
keywords: Accuracy; Reproducibility; ncRNA