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

作   者：唐芳,楚立威,舒文波,贺学娇,王丽娟,卢孟柱
期刊名称：Plant Methods
影响因子：3.170
卷 期 号：15
页     码：无
关键词：内参基因筛选
论文摘要：
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.