Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina

Abstract

Arabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by reverse-transcription quantitative Real-Time PCR (RT-qPCR). For the normalisation of expression data in RT-qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A . alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3 , HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3 , PSB33 and EIF4a for heat; CAC , TUA5 , ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize RT-qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality RT-qPCR data in A . alpina .

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

“…Liu et al 27 analyzed perennial ryegrass under high-temperature stress and identified HIS3 and eIF4A as the most suitable reference genes. In another study, EF-1α and UBQ were detected as the most stable internal reference genes in potato and Arabis alpina under drought conditions 28 , 29 . Therefore, verifying the expression stability of reference genes under different experimental conditions is critical when selecting reference genes to standardize gene expression levels.…”

Evolutionary Comparison of the Developmental/Physiological Phenotype and the Molecular Behavior of SPIRRIG Between Arabidopsis thaliana and Arabis alpina

“…(C) The expression levels of AaSPI were determined in the above ground parts of seedlings of pep1 , Aaspi-1 , Aaspi-2 , and Aaspi-3 by qPCR. Normalization was carried out using AaTUA5 and AaCAC ( Stephan et al, 2019 ).…”

“…Higher dilutions were not possible due to the concentration of RNA/cDNA. Primers for reference genes were described before ( Wang et al, 2009 ; Stephan et al, 2019 ). Primers for genes of interest were accepted with an efficiency of 80–120% and a correlation between −1 and −0.99 ( Supplementary Table S1 ).…”

Selection and validation of experimental condition-specific reference genes for qRT-PCR in Metopolophium dirhodum (Walker) (Hemiptera: Aphididae)

“…Nevertheless, gene expression analyses are affected by many factors such as the quality of RNA samples, the efficiency of reverse transcription, and PCR efficiency 2 , 3 . For accurate comparisons of expression levels, the expression data of the genes of interest are normalized against the expression data for a reference gene 4 . Moreover, the reference gene compensates for the above-mentioned limitations 5 .…”