title: Normalization for Relative Quantification of mRNA and microRNA in Soybean Exposed to Various Abiotic Stresses.
abstract: Plant microRNAs are small non-coding, endogenic RNA molecule (containing 20-24 nucleotides) produced from miRNA precursors (pri-miRNA and pre-miRNA). Evidence suggests that up and down regulation of the miRNA targets the mRNA genes involved in resistance against biotic and abiotic stresses. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful technique to analyze variations in mRNA levels. Normalizing the data using reference genes is essential for the analysis of reliable RT-qPCR data. In this study, two groups of candidate reference mRNAs and miRNAs in soybean leaves and roots treated with various abiotic stresses (PEG-simulated drought, salinity, alkalinity, salinity+alkalinity, and abscisic acid) were analyzed by RT-qPCR. We analyzed the most appropriate reference mRNA/miRNAs using the geNorm, NormFinder, and BestKeeper algorithms. According to the results, Act and EF1b were the most suitable reference mRNAs in leaf and root samples, for mRNA and miRNA precursor data normalization. The most suitable reference miRNAs found in leaf and root samples were 166a and 167a for mature miRNA data normalization. Hence the best combinations of reference mRNAs for mRNA and miRNA precursor data normalization were EF1a + Act or EF1b + Act in leaf samples, and EF1a + EF1b or 60s + EF1b in root samples. For mature miRNA data normalization, the most suitable combinations of reference miRNAs were 166a + 167d in leaf samples, and 171a + 156a or 167a + 171a in root samples. We identified potential reference mRNA/miRNAs for accurate RT-qPCR data normalization for mature miRNA, miRNA precursors, and their targeted mRNAs. Our results promote miRNA-based studies on soybean plants exposed to abiotic stress conditions.
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Weican
authors.initials: W
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Deng
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Chen
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Dong
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Aysha
authors.initials: A
authors.lastname: Jameel
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: He
authors.initials: H
authors.lastname: Yang
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Fawei
authors.initials: F
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, Jilin, 130118, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Li
mesh_terms: D000465:Algorithms; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012984:Software; D013025:Glycine max / Q000235:genetics*; D013312:Stress, Physiological / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D018749:RNA, Plant