Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT), Elongation factor 1-alpha (EF1-α), Eukaryotic translation initiation factor 3E (ETIF3E), alpha tubulin (α-TUB), beta tubulin (β-TUB), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin (CYP), Clathrin adaptor complex (CAC), Serine/threonine-protein phosphatase 2A (PP2A), FtsH protease (FtsH), 18S ribosomal RNA (18S rRNA), S-adenosyl methionine decarboxylase (SAMDC) and Rubisco activase (RCA) and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1), a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis

“…The total span of quantification cycle (Cq) values across all samples were much smaller for the candidate reference genes than for the haustorium-related genes, Cr - PX - 2 and Cr - XTH - 1 (Additional file 3). This was expected as the candidate reference genes were selected based on earlier reports showing relatively stable expression of similarly annotated genes in other plant species [26–28]. To more precisely compare the expression stabilities of the five candidate reference genes, the NormFinder algorithm was used to calculate the stability value [29], and the coefficient of variation CV and the stability parameter M were calculated as described by Hellemans et al [30].…”

Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation

“…Whereas, a V value cutoff of 0.15 is accepted to determine the optimal number of RGs. Since our samples are heterogeneous, M values below 1.00 were considered as stable (Gopalam et al 2017;Zhou et al 2017).…”

“…The second point is often neglected (Die and Román 2012), and for the third and probably most critical point, typically a single housekeeping gene is considered for normalization. Such genes are involved in fundamental cellular processes and therefore expected to be ubiquitously and constitutively expressed across various biological and ontogenetic conditions, independent of the experimental set-up (Coker and Davies 2003;Gopalam et al 2017;Gutierrez et al 2008;Li et al 2011). Though, multiple studies have questioned their suitability for normalization and have stressed the importance of proper RG selection and systematic validation of candidate genes specifically for the experimental condition under study (Czechowski et al 2005;Dekkers et al, 2012;Gopalam et al, 2017;Guénin et al 2009;Gutierrez et al 2008;Kozera and Rapacz 2013;Radonić et al 2004).…”

“…Such genes are involved in fundamental cellular processes and therefore expected to be ubiquitously and constitutively expressed across various biological and ontogenetic conditions, independent of the experimental set-up (Coker and Davies 2003;Gopalam et al 2017;Gutierrez et al 2008;Li et al 2011). Though, multiple studies have questioned their suitability for normalization and have stressed the importance of proper RG selection and systematic validation of candidate genes specifically for the experimental condition under study (Czechowski et al 2005;Dekkers et al, 2012;Gopalam et al, 2017;Guénin et al 2009;Gutierrez et al 2008;Kozera and Rapacz 2013;Radonić et al 2004). The practice of using single popular housekeeping gene, e.g., actin (ACT), glyceraldehyde 3-phophate dehydrogenase (GAPDH), ubiquitin (UBQ), and rRNA subunits, have already led to erroneous and unreliable normalization of (RT)-qPCR results (Gutierrez et al 2008;Kozera and Rapacz 2013;Vandesompele et al 2002;Yim et al 2015).…”

De novo Sequencing and Analysis of Salvia hispanica Tissue-Specific Transcriptome and Identification of Genes Involved in Terpenoid Biosynthesis

“…qPCR primers were designed using the online Primer 3 software (Additional file 1). The housekeeping genes Serine/threonine-protein phosphatase 2A (PP2A) and Cyclophilin (CYP) were used as internal controls to normalize the data [37]. Three biological replicates were used.…”