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

PloS One
Rahul GopalamAjay W Tumaney

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 th...Continue Reading

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Methods Mentioned

BETA
reverse transcription PCR
protein folding
electrophoresis
PCR
acylation

Software Mentioned

TBLASTN
BLAST
Excel
Normfinder
Primer
geNorm
qBaseplus
Primer3Plus

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