Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.
IR@CFTRI: CSIR-Central Food Technological Research Institute, Mysore
View Archive Info| Field | Value | |
| Relation |
http://ir.cftri.com/13326/
https://doi.org/10.1371/journal.pone.0186978 |
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| Title |
Selection and validation of appropriate
reference genes for quantitative real-time PCR
analysis in Salvia hispanica.
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| Creator |
Gopalam, R.
Sunny, D. Rupwate Ajay, W. Tumaney |
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| Subject |
03 Biochemistry & Molecular Biology
01 Oilseeds |
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| Description |
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.
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| Date |
2017
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| Type |
Article
PeerReviewed |
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| Format |
pdf
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| Language |
en
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| Identifier |
http://ir.cftri.com/13326/1/PLoS%20ONE%2012%2811%29.pdf
Gopalam, R. and Sunny, D. Rupwate and Ajay, W. Tumaney (2017) Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica. PLOS One. pp. 1-17. |
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