Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments

Abstract

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala.Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

“…These expression stability differences might be a result of different species/cultivars analyzed in the compared experiment conditions. It is consistent with the previous studies that the stability of reference genes is not only cultivar/species specific but may also be tissue specific and influenced by the different experimental treatments (Nicot et al, 2005 ; Štajner et al, 2013 ; Zhuang et al, 2015 ). This is also the primary reason for validating the stability of reference genes for a specific genotype and/or experimental treatment.…”

Reference Gene Selection for RT-qPCR Analysis of Flower Development in Chrysanthemum morifolium and Chrysanthemum lavandulifolium

“…The optimal reference genes were determined by synthesizing using the above described methods. GeNorm and NormFinder are the most widely used algorithms, and they were different from each other in many studies, such those in as flax (Huis et al, 2010 ), rubber tree, tobacco (Schmidt and Delaney, 2010 ), and Oxytropis ochrocephala Bunge (Zhuang et al, 2015 ). However, there are still some studies in which these algorithms showed very few differences from each other, such as those in coffee (Cruz et al, 2009 ) and rice (Jain, 2009 ).…”

Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data

“…Taking into account the discrepancy on stability analysis among three different analytical programs, RefFinder program was applied for reanalyzing the 15 reference genes based on the geometric mean of their attributed weight, which used geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to rank and compare candidate reference genes [41] ( Table 4). e ranking order of the top five most stable and unstable reference genes obtained by RefFinder was broadly in consistent with the results provided by geNorm and NormFinder and had slight differences with the results from BestKeeper.…”