Abstract
BackgroundQuantitative 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Given its high sensitivity, quantitative accuracy, low cost and specificity, quantitative reverse transcriptase polymerase chain reaction has become theTang et al Plant Methods (2019) 15:35 experimental condition in different tissues or species [5]
Verification of amplification and efficiency of the primers A total of 12 protein-coding genes and 6 small noncoding RNAs were used as candidate reference genes for quantitative detection of miRNAs and mRNAs
The PCR amplification specificities were confirmed by melting curves (Additional file 1: Fig. S1), agarose gel electrophoresis (Additional file 1: Fig. S2) and sequencing (Additional file 2: Fig. S3), which demonstrated the specific product of expected size and sequence
Summary
Quantitative accuracy, low cost and specificity, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) has become theTang et al Plant Methods (2019) 15:35 experimental condition in different tissues or species [5]. 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 [17,18,19,20]. MRNAs and miRNAs could be 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 important to select a suitable reference gene for normalization in quantitative PCR of miRNA and mRNA. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. It is important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA
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