An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development
By K. E. Reid, Niclas Olsson, J. Schlosser, Fred Peng, Steven T. Lund. BMC Plant Biology, 2006
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BackgroundAccuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents.ResultsWe describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, α-tubulin, β-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series.ConclusionIn order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-α and SAND as the most relevant reference genes for this purpose.
Subfields: Genetics, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/234a09c639df44c63a6fa9860bd267cb099f231b
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Citations in the corpus
- Drought-induced changes in development and function of grapevine (Vitis spp.) organs and in their hydraulic and non-hydraulic interactions at the whole-plant level: a physiological and molecular update (2010, methodology): “Grapevine aquaporins have been cloned by Baiges et al. (2001), Picaud et al. (2003), Perrone et al. (2006), Reid et al. (2006), Galmés et al. (2007), Fouquet et al. (2008), Glissant et al. (2008), Schlosser et al. (2008), Shelden et al. (2009) and Vandeleur et al. (2009), and reported to be…”
- Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus (2011, neutral): “, 2011), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and elongation factor 1-α (EF1-α) (Reid et al., 2006), were considered for use in qRT-PCR after analysis of gene expression stability in the geNorm software (Vandesompele et al.”
- Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR (2009, background): “The importance of reference genes for plant qPCR analysis has been recently emphasized, even though the identification of these genes is quite laborious (Brunner et al. 2004; Reid et al. 2006 ).”
- Identification of Four Soybean Reference Genes for Gene Expression Normalization (2008, background): “, 2005), GAPDH, actin and EF1-α in grape (Vitis spp.; Reid et al., 2006), ubiquitin in poplar (Populus spp.”