Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo

Abstract

Expression of ACT, EF1A; H2A, EF1A, ACT and 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.

Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses

“…The least stable genes are on the left and the most stable genes on the right. Total leaves and roots of all stress treatments, SR and SL salt-treated roots and leaves, PR and PL PEG-treated roots and leaves, CR and CL cold-treated roots and leaves, HR and HL heat-treated roots and leaves Table 2 Most stable and least stable combination of reference genes based on RefFinder analysis Experimental treatments ACT were identified as stable reference genes in black gram (Vigna mungo) (Kundu et al 2013), whereas they exhibited unstable expression under four abiotic stresses (osmotic, salt, cold, and heat) in Caragana intermedia (Zhu et al 2013). In this study, ACT could be applied as a stable reference gene only for salinity-stressed leaves and TUB was not suitable for gene normalization in leaves or roots of creeping bentgrass (Table 2).…”

“…Quantitative real-time PCR (qRT-PCR) is a commonly used analysis used to confirm gene expression patterns identified through transcriptomic profiling. However, accurate determination of candidate gene expression levels and patterns depends on the use of stable internal reference genes (RGs) (Chen et al 2014;Jain et al 2006;Kundu et al 2013;Li et al 2012;Pfaffl 2001;Wang et al 2014a). Several RGs such as actin (ACT), tubulin (TUB), elongation factor 1a (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18s ribosomal RNA (18S rRNA) involved in primary metabolism or other cellular processes were commonly utilized in previous studies and exhibited either stable and constitutive expression at certain developmental stages in specific tissues or responding to experimental stimuli Li et al 2014) but were unstable under other experimental conditions (Chen et al 2014;Huang et al 2014;Zhu et al 2013).…”

Reference gene selection for gene expression studies in lily using quantitative real-time PCR

“…Under drought stress, UBQ7, EF-1a8, TUA10, and GAPDH showed less gene-expression variation in both the leaves and the roots (Wang et al, 2013). For Vigna mungo, a high stability was obtained for ACT and EF-1a during mungbean yellow mosaic India virus stress; H2A, EF-1a, and ACT were found to be most suitable under salinity stress experiments; and TUB and 18S (18S rRNA) during drought treatments (Kundu et al, 2013). Commonly known housekeeping genes like GAPDH, ACTB, and 18S have been used in many expression studies for different tissues (Thellin et al, 1999;Bustin, 2000).…”

Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress

“…To compare the effects of salt stress and drought stress on statistic reference genes, we screened eight studies (Chang et al 2012;Dombrowski and Martin 2009;Kumar et al 2013;Kundu et al 2013;Lee et al 2010a;Li et al 2012;Park et al 2012;Zhu et al 2013) that applied candidate RGs under salt and drought stress in the same species. As shown in Table S3, 200-250 mM NaCl solution is commonly used to generate salt stress, while 10-40 % PEG is commonly used to create drought stress.…”