Reference genes assessment for the seagrass Posidonia oceanica in different salinity, pH and light conditions

Abstract

The stability on the expression level of putative reference genes (RGs) for reverse transcriptase-quantitative PCR (RT-qPCR) was tested in the seagrass Posidonia oceanica for plants collected in different environmental conditions (at different depths and in different salinity and pH). Besides adult plants, seedlings were also used for pH and salinity experiments. The putative RGs encompassed the most frequently used in other species to normalize RT-qPCR in a range of experimental conditions. Assignment of the best RGs has been performed with three Excelbased applets, BestKeeper, geNorm and NormFinder, and the results showed that best RGs may change depending on the experimental conditions tested, plant age and on the software utilized. Preferred sets of RGs are proposed for each of the conditions and a general procedure concerning their selection is discussed. In addition, expression levels of four genes involved in plant response to light, salinity and pH variation have also been evaluated.

Response of the seagrass Posidonia oceanica to different light environments: Insights from a combined molecular and photo-physiological study

“…Relative gene expression in plants collected in the shallow stand was analyzed using samples collected in the deep stand as control. L23 (GenBank: GO347779), EF1A (GenBank: GO346663) and NTUBC2 (GenBank: GO347619) were used as reference genes (RGs) for the relative quantification because they have been previously identified as the most stable RGs in P. oceanica in the same experimental conditions (Serra et al, 2012).…”

“…For a detailed description, see Serra et al (2012). Amplification efficiency (E) for all primer pairs has been calculated from the slopes of standard curves of the threshold cycle (Ct) vs. cDNA concentration, with the equation E ¼ 10 À1/slope À1.…”

“…The recent development of molecular resources for different seagrass species (e.g. Franssen et al, 2011;Wissler et al, 2009) has greatly facilitated the investigation of differentially expressed genes in response to environmental variations (Bergmann et al, 2010;Bruno et al, 2010;Gu et al, 2012;Massa et al, 2011;Reusch et al, 2008;Serra et al, 2012;Winters et al, 2011), and first attempts have been already carried out in Posidonia oceanica, to couple genomic and eco-physiological approaches (Dattolo et al, 2013;Mazzuca et al, 2013;Procaccini et al, 2012).…”

Response of key stress-related genes of the seagrass <i>Posidonia oceanica</i> in the vicinity of submarine volcanic vents

“…The screened panel included the eukaryotic initiation factor-4A (eIF4A) (F 5'-TTCTGCAAGGGTCTTGACGT-3' and R 5'-TCACACCCAAGTAGTCACCAAG-3'; E = 1.85; R 2 = 0.99) as well as the ones already published in Serra et al (2012): ubiquitin (UBI), ribosomal protein L23 (L23), elongation factor 1-alpha (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal RNA 18S (18S) and ubiquitin-conjugating enzyme (NTUBC2). Three different algorithms were utilized to identify the best RGs in our experimental design: BestKeeper (Pfaffl et al, 2004), geNorm (Vandesompele et al, 2002) and NormFinder (Andersen et al, 2004).…”

“…Table 1 lists selected GOI, their functions, primers' sequences and amplicon sizes. Primers were optimized as in Serra et al (2012). The sequences are deposited in GenBank under the accession numbers shown in Table 1.…”