Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.)

Abstract

BackgroundQuantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L).ResultsSpecific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.ConclusionsThe use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis

“…Thus, it is understandable that the top reference genes ranked by the two programs were different. Discrepancies between geNorm and NormFinder have also been noted in previous studies (Hong et al, 2008;Wan et al, 2010;Huis et al, 2010). …”

Selection of Endogenous Reference Genes for Gene Expression Analysis in the Mediterranean Species of the <I>Bemisia tabaci</I> (Hemiptera: Aleyrodidae) Complex

“…DisCussion qRt-pcR has proved to be an accurate and sensitive method for gene expression analysis (andersen et al 2004;Willems et al 2006;Hoogewijs et al 2008;Langnaese et al 2008;tatsumi et al 2008;Bustin et al 2009;Huis et al 2010) and to study developmental processes in biological systems (Exposito-Rodriguez et al 2008). Rt-pcR data analysis is greatly influenced by the selection of reference genes (Bustin 2002;vandesompele et al 2002;Gutierrez et al 2008), and the lack of stable reference genes can create great risks of misinterpretation of results (perez et al 2008).…”

Effects of Water Stress on Germination and Growth of Linseed Seedlings (Linum usitatissimum L), Photosynthetic Efficiency and Accumulation of Metabolites

“…It is one of the earliest cultivated plants known and, since ancient times, it has been grown widely for its high-quality, cellulose-rich, bast fibers and also for its oil (Zohary & Hopf, 2004;Huis et al, 2010). The oil of linseed is an excellent nutritional supplement being rich in α-linolenic acid and omega-3 fatty acid.…”

“…The oil is also used in the production of industrial raw materials (Vaisey-Genser & Morris, 2003;Foster et al, 2009;McKenzie & Deyholos, 2011). In the textile industry, the fibers of linseed have been largely replaced by cotton or by synthetic fibers but they are still used in the high-quality linen materials and are also now being incorporated in the polymeric matrix of biocomposites to improve their mechanical properties (Chemikosova et al, 2006;Bodros et al, 2007;Huis et al, 2010). In 2009, the top producers of linseed were Canada, India and China, with 45% of world production being in Canada (FAO, 2009).…”