An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

BMC Plant Biology BioMed Central Methodology article Open Access An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development Karen E Reid, Niclas Olsson, James Schlosser, Fred Peng and Steven T Lund* Address: Faculty of Land and Food Systems, University of British Columbia, Vancouver, Canada Email: Karen E Reid - ; Niclas Olsson - ; James Schlosser - ; Fred Peng - ; Steven T Lund* - * Corresponding author Published: 14 November 2006 BMC Plant Biology 2006, 6:27 doi:10.1186/1471-2229-6-27 Received: 19 July 2006 Accepted: 14 November 2006 This article is available from: http://www.biomedcentral.com/1471-2229/6/27 © 2006 Reid et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Accuracy 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. Results: We 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. Conclusion: In order to quantify relative transcript abundances accurately using real-time RTPCR, 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. Page 1 of 11 (page number not for citation purposes) BMC Plant Biology 2006, 6:27 Background Transcriptomics is an important growing field of molecular biology. Gene expression analyses are increasing our understanding of signalling and metabolic pathways underlying developmental and cellular processes. Realtime RT-PCR is currently one of the more powerful and sensitive techniques for analyzing gene expression. It provides outstanding accuracy of RNA quantification and has a broad dynamic range over wide experimental conditions [1-7]. As in other expression studies, data normalization is essential to control for experimental error introduced throughout sample preparation. It has been shown that real-time RT-PCR results are highly dependent on the reference genes chosen [8], which supports putting considerable effort into validating gene(s) chosen for normalization prior to extensive experimentation. Useful reference genes must not only be present in all samples but the expression levels need to remain constant relative to experimental pressures introduced. Data normalization can be problematic and several strategies have been reviewed [9]. Housekeeping genes are constitutively expressed and required for cellular survival, including functions such as cell wall structure and primary metabolism. Previously, these have been found to be reasonable internal reference genes for normalizing real-time data. These genes are expected to exhibit minor differences in their expression profiles under diverse experimental conditions. Examples such as GAPDH, 18S rRNA and EF1-α have been widely used in RNA blot analyses and are commonly used for real-time RT-PCR in various plant species [2,3,6,7,10,11]. While these genes have been found to be appropriate for some experiments, other candidates were recently reported to outperform these classical ones [12]. Grape berries undergo significant metabolic changes throughout their development, orchestrated in part by the up and down regulation of transcripts. This development follows a double sigmoidal pattern characterized by two periods of cellular expansion separated by a period of slowed growth [13]. The ability to identify transcripts that are resistant to growth fluctuations or stresses is challenging; therefore, it is important to identify candidate reference genes that are subject to only minimal regulation during an individual experiment, permitting accurate transcriptional analyses. To date, a limited number of real-time RT-PCR experiments focusing on grape berries has been published. Based on microarray and real-time RT-PCR data, UBQ-L40 [14] and one paralog of EF1-α [2] were previously reported as being stably expressed in grape berries. Prior to evaluating expression patterns in biological samples, it is important to ensure that the RNA being used for http://www.biomedcentral.com/1471-2229/6/27 cDNA synthesis is pure and not degraded. Grapevine tissues, like those in many higher plant species, contain abundant polyphenolic and polysaccharide compounds which cause challenges when isolating RNA. At full maturity, for example, Cabernet Sauvignon berries contain approximately 26 percent soluble solids, mainly glucose and fructose, and these sugars can co-precipitate with nucleic acids into a viscous gelatin-like pellet during RNA isolation. Moreover, due to the low RNA content in the maturing berries, success is limited in capturing low concentrations of nucleic acids using large-volume extraction protocols. In this study, we present an RNA isolation protocol adapted from a previously described procedure developed for the evergreen tree, Cinnamomum tenuipilum [15]. Our protocol compensates for the acidic nature of grape berries and introduces modifications to both increase RNA yield and minimize contaminating polysaccharides. We demonstrate that high quality and quantity of RNA can be obtained from grape berries from all develop (...truncated)