Selection and validation of reference genes for quantitative expression analysis of miRNAs and mRNAs in Poplar

(2019) 15:35 Tang et al. Plant Methods https://doi.org/10.1186/s13007-019-0420-1 Plant Methods Open Access RESEARCH Selection and validation of reference genes for quantitative expression analysis of miRNAs and mRNAs in Poplar Fang Tang1,2†, Liwei Chu1†, Wenbo Shu1,3, Xuejiao He1, Lijuan Wang1,2 and Mengzhu Lu1,2* Abstract Background: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. However, because of the specially designated reverse transcription and shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA expression in plants, and different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA. Results: In this study, a modified reverse transcription PCR protocol was adopted for selecting and validating universal internal reference genes of mRNAs and miRNAs. Eight commonly used reference genes, four stably expressed novel genes in Populus tremula, three small noncoding RNAs and three conserved miRNAs were selected as candidate genes, and the stability of their expression was examined across a set of 38 tissue samples from four developmental stages of poplar clone 84K (Populus alba × Populus glandulosa). The expression stability of these candidate genes was evaluated systematically by four algorithms: geNorm, NormFinder, Bestkeeper and DeltaCt. The results showed that Eukaryotic initiation factor 4A III (EIF4A) and U6-2 were suitable for samples of the callus stage; U6-1 and U6-2 were best for the seedling stage; Protein phosphatase 2A-2 (PP2A-2) and U6-1 were best for the plant stage; and Protein phosphatase 2A-2 (PP2A-2) and Oligouridylate binding protein 1B (UBP) were the best reference genes in the adventitious root (AR) regeneration stage. Conclusions: The purpose of this study was to identify the most appropriate reference genes for qRT-PCR of miRNAs and mRNAs in different tissues at several developmental stages in poplar. U6-1, EIF4A and PP2A-2 were the three most appropriate reference genes for qRT-PCR normalization of miRNAs and mRNAs during the plant regeneration process, and PP2A-2 and UBP represent the best reference genes in the AR regeneration stage of poplar. This work will benefit future studies of expression and function analysis of miRNAs and their target genes in poplar. Keywords: Reference genes, MicroRNAs, mRNAs, qRT-PCR, Normalization, Development, Poplar Introduction Given its high sensitivity, quantitative accuracy, low cost and specificity, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) has become the *Correspondence: † Fang Tang and Liwei Chu contributed equally to this work 1 State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China Full list of author information is available at the end of the article most common and widely used technique for quantifying miRNA expression and mRNA transcript levels among different tissues and experimental conditions in plants [1, 2]. However, the accuracy of qRT-PCR is easily affected by several factors, including the quality of RNA samples, reverse transcription efficiency, cDNA quality and amount, and differences in extraneous tissue and cell activities [2–4]. To avoid bias in qRT-PCR analysis, validation of suitable reference genes for data normalization is an elementary prerequisite for each © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tang et al. Plant Methods (2019) 15:35 experimental condition in different tissues or species [5]. However, no single reference gene can be universal under all experimental situations, even including the most stable reference gene(s) reported [6, 7]. Therefore, optimal reference genes should be validated for different species, tissues or specific treatments. MicroRNAs (miRNAs) are endogenous ~ 22 nt small noncoding RNAs that guide the cleavage or repress the translation of their target mRNAs by approximate basepairing rules [8, 9] or mediate mRNA decay by directing rapid deadenylation of mRNAs [10]. In plants, miRNAs are master regulators in controlling developmental processes and in response to biotic and abiotic stress responses [11–15]. Due to its short sequence (only ~ 22 nt in length), the quantification of miRNAs by qRT-PCR requires extending the length of mature miRNAs using stem-loop primers [7, 16] or adding poly(A)-tails [17– 20]. This extension requirement causes different internal reference genes to be commonly used for normalization in qRT-PCR of miRNAs and mRNAs. Some housekeeping genes, such as actin 7 (ACT 7), eukaryotic initiation factor 4A III (EIF4A), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A-2 (PP2A-2), were widely adopted for gene expression analysis in diverse plants as reference genes [21–23]. Several noncoding RNA and small RNA, such as 5.8S ribosomal RNA (5.8S rRNA) and U6 small nuclear RNA (U6 snRNA), are commonly used as reference genes for miRNA quantity [24, 25]. Hurteau developed a modified universal reverse transcription PCR protocol, in which mature miRNAs could be polyadenylated by poly (A) polymerase and reverse transcribed into cDNA using oligo-dT primers [17]. Then, mRNAs and miRNAs could be specifically amplified and quantified at same transcriptional level, and the relative quantification of a miRNA and its predicted mRNA target can be both assessed precisely [17]. In this case, it is particularly important to select a suitable reference gene for normalization in quantitative PCR of miRNA and mRNA. As a typical model woody plant, Populus has many advantages in basic research, such as rapid and perennial growth, moderate genome size, biomass-related traits and facile transformation [26]. Completion of the genomic sequence for Populus trichocarpa (black cottonwood) [27] has led to the development of genomic and molecular resources, and the ideal genetic transformation system provides a powerful genetic analysis tool for dissecting adaptive traits in poplar [28, 29]. Poplar c (...truncated)