Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp.

Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

“…3.1 Characterization of the expression profiling of candidate reference genes Gene selection was based on similar studies conducted in other plant species subjected to abiotic stresses, including salinity (Goulao et al 2012;Kandu et al 2013;Guo et al 2014;Xiao et al 2015). The ten candidates [Ubi (Ubiquitin), Tub (Tubulin), S23 (40S ribosomal protein), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), Elf-4a (Eukaryotic initiation factor 4α), Ef-1 (Elongation factor 1α), Ap47 (Clathrin adaptor complexes subunit), Apt (Adenine phosphoribosyltransferase), Act (Actin) and PP2A (Serine threonine protein phosphatase)] complied with the PCR requirements showing a single peak in the dissociation curve and efficiencies ranging between 84 and 104 % ( Table 1).…”

“…Based on the available sequences of C. glauca database (Hocher et al 2011), a set of ten candidate genes were selected, comprising several commonly used reference genes in plants (Remans et al 2014;Goulao et al 2012;Gutierrez et al 2008): ubiquitin (Ubi); adenine phosphoribosyltransferase (Apt); elongation factor 1α (Ef-1); tubulin (Tub); eukaryotic initiation factor 4α (Elf-4A); glyceraldehydes 3-phosphate dehydrogenase (GAPDH); 40S ribosomal protein (S23); clathrin adaptor complexes subunit (Ap47); actin (Act); serine/threonine protein phosphatase (PP2A) ( Table 1). For each gene, specific primers were designed using the Primer3 software (Rozen and Skaletsky 2000), according to the following parameters: melting temperature (Tm) of 58-62°C, GC content of 45-55 % and length of the primers between 20 and 22 bp.…”

“…To confirm the reliability of the potential reference genes, the transcriptional activities of C.glauca citrate synthase (CgCs) and ascorbate peroxidase (CgAPX) along the imposed stress (200, 400 and 600 mM) were estimated in relation to their respective controls (Goulao et al 2012), using the three most-versus the three least stable reference genes for data normalization. The qRT-PCR amplification conditions were the same as described above.…”

“…These must be expressed at rather constant levels among the samples under study. Traditionally, reference genes are selected among housekeeping genes, which are important for basic cellular processes and therefore assumed to be constantly expressed independently of the plant organ or experimental conditions (Goulao et al 2012). However, there are evidences that the mRNA levels of these genes are not always constant, particularly in the case of stress conditions (Barber et al 2005;Gutierrez et al 2008).…”

Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress

“…Statistic analysis of reference gene stability under drought stressFor the analysis of reference genes under drought stress, 27 publications within 26 plant species were collected in Table S2(Bin et al 2012;Borges et al 2012;Carvalho et al 2010;Chandna et al 2012;Chang et al 2012;Cruz et al 2009;Dombrowski and Martin 2009;Dong et al 2012;Garg et al 2010;Goulao et al 2012;Hong et al 2008;Janska et al 2013;Kumar et al 2013;Kundu et al 2013;Le et al 2012; Lee et al 2010a, b;Maksup et al 2013;Park et al 2012;Qi et al 2010;Reddy et al 2013;Schmidt and Delaney 2010;Shi et al 2012;Wan et al 2010;Wei et al 2013;Zhang et al 2013;Zhu et al 2013).…”