Abstract
To accurately evaluate expression levels of target genes, stable internal reference genes is required for normalization of quantitative real-time PCR (qRT-PCR) data. However, there have been no systematical investigation on the stability of reference genes used in the bedstraw weed, Galium aparine L. (BGA). In this study, the expression profiles of seven traditionally used reference genes, namely 18S, 28S, ACT, GAPDH, EF1α, RPL7 and TBP in BGA were assessed under both biotic (developmental time and tissue), and abiotic (temperature, regions and herbicide) conditions. Four analytical algorithms (geNorm, Normfinder, BestKeeper and the ΔCt method) were used to analyze the suitability of these genes as internal reference genes. RefFinder, a comprehensive analytical software, was used to rank the overall stability of the candidate genes. The optimal normalization internal control genes were ranked as: 28S and RPL7 were best for all the different experimental conditions (developmental stages, tissues, temperature, regions and herbicide treatment); 28S and RPL7 for developmental stages; TBP and GAPDH for different tissues; 28S and GAPDH were relatively stable for different temperature; 28S and TBP were suitable for herbicide treatment. A specific set of reference genes were recommended for each experimental condition in BGA.
Highlights
Quantitative Real-time PCR is considered as the most common method for gene expression quantification, due to high sensitivity and efficiency[1,2,3]
The expression levels of reference genes have been commonly quantified in various weeds by Quantitative Real-time PCR (qRT-PCR)[15,16]. One reference gene such as 18S, glyceraldehde-3-phosphate dehydrogense (GAPDH), or ACT was selected for normalization the target gene expression by qRT-PCR under all the diverse experimental conditions in multiple studies
Our results revealed that specific reference genes were required for qRT-PCR normalization under various experimental conditions
Summary
Quantitative Real-time PCR (qRT-PCR) is considered as the most common method for gene expression quantification, due to high sensitivity and efficiency[1,2,3]. One reference gene such as 18S, GAPDH, or ACT was selected for normalization the target gene expression by qRT-PCR under all the diverse experimental conditions in multiple studies. Seven common reference genes, such as 18S, 28S, ACT, GAPDH, EF1α, RPL7 and TBP, were selected and evaluated for target gene expression normalization using the qRT-PCR method in BGA.
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