Abstract
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.
Highlights
Quantitative reverse transcription polymerase chain reaction is an efficient, sensitive and reliable technique for quantifying the expression profiles of target genes in different tissues, following different hormone treatments and under various stresses [1]. qRT-PCR enables comparison of changes in the expression of a target gene to changes in reference genes, including abundant transcripts and low-abundance transcripts of the target gene
Our results provide a foundation for other researchers to choose reference genes for the normalization of mRNA levels by qRT-PCR in this tree species
TUB is a member of the tubulin gene family, which are commonly used as reference genes [45] but in Metasequoia, its expression stability was low in different tissues and hormone treatment
Summary
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an efficient, sensitive and reliable technique for quantifying the expression profiles of target genes in different tissues, following different hormone treatments and under various stresses [1]. qRT-PCR enables comparison of changes in the expression of a target gene to changes in reference genes, including abundant transcripts and low-abundance transcripts of the target gene. Normalization requires the selection of one or more reference genes showing constant and stable expression levels in different tissues and following different treatments [4,5]. To identify an accurate and efficient target gene expression profile for gene expression analysis, we determined the best reference genes in Metasequoia for studying different plant tissues and following different hormone treatments before normalization of gene expression. Several bioinformatics tools including geNorm [22], NormFinder [23] and BestKeeper [24] have been utilized to analyze and assess the expression stability of reference genes for qRT-PCR data normalization. BestKeeper is an Excel-based tool for selecting the best candidate using pairwise correlations In addition to these methods, the ∆Ct [25] and GrayNorm [26] algorithms have been widely employed for data analysis. Our results provide a foundation for other researchers to choose reference genes for the normalization of mRNA levels by qRT-PCR in this tree species
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