Computational protocol: Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

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Protocol publication

[…] The 60S, GAPDH, UBQ, ZTL, TEF1, peroxidase (POD), and superoxide dismutase (SOD) orthologous genes of wheat were used as ‘query’ to BLAST against available perennial ryegrass expressed sequence tags in the GenBank database (NCBI). All genes were named based on their similarity to known genes with sequence similarity from 90% to 98%. Primer sequences for an additional six reference genes’ were identified from a previous study .Primers were designed using Primer 3 ( using the following criteria: Tm between 58–62°C (optimum Tm of 60°C); PCR product size between 75–200 base pairs, length of 18 to 24 nucleotides in optimal length of 20 nucleotides) and GC content from 40% to 60%. In order to check the primers’ specificity, the PCR products were analyzed using 2.0% agarose gel electrophoresis and stained using Gelred (Biotium, USA).QRT-PCR reactions were executed in 96-well blocks with a BIO-RAD CFX96 Real-Time PCR system (Bio-Rad, USA) using SYBR Premix Ex TaqTM (TaKaRa, Japan). The PCR reactions were 20 μl in volume and contained 10 μl 2×SYBR Premix Ex TaqTM, 2 μl diluted cDNA reaction mixture, 0.4 μl ROX Reference Dye II, and 1 μl 10 μM for each primer. The cycling conditions were as recommended by the manufacturer: 5 min at 95°C, 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. At the end of the cycling process, the temperature was raised from 60°C to 95°C to obtain the dissociation curve. Three technical replicates were performed for each biological replicate – gene combination. The final cycle threshold (CT) values were calculated as the means of all nine values. [...] The stability of reference gene expression was analyzed using four different VBA (Visual Basic Applet) applets: Delta CT method , geNorm (ver. 3.5) , BestKeeper (ver. 1.0) and Normfinder (ver. 0.953) . Results from CFX manager (Bio Rad) were exported into Microsoft Excel 2007 and transformed to create input files for each target according to the requirements of each software. The geNorm tool ranked the reference genes by calculating the gene expression stability value (M1) based on the average pairwise expression ratio. The most stable reference gene has the lowest M1 value, while the least stable one presents the highest M1 value. The program considers M1 values below 1.5 to indicate stable expression. Normfinder is a Microsoft Excel application which ranks candidate genes according to stability index M2 based on the average pairwise variation of a given gene compared to all other studied genes . The more stably expressed genes exhibit lower M2 values. BestKeeper is another Excel-based tool that identifies the most stably expressed genes by making comparisons of the coefficient of variance (CV) and the standard deviation (SD) . The most stable genes are those with the lowest coefficient of variance and standard deviation (CV ± SD). Finally, the reference genes’ stability values derived from each of the three software tools were then used to create a comprehensive rank each of the 11 candidate reference genes in order from most to least stable expression under each abiotic stress condition using RefFinder . This is a user-friendly, web-based tool developed for comprehensively evaluating and screening reference genes from extensive experimental datasets .Gene-specific PCR efficiency was also calculated based on standard curves using a 10-fold serial dilution of the mixed cDNA template for each primer pair. The correlation coefficients (R2) and slope values were acquired from the standard curve. Calculate the coefficient of variation (CV) according to the equation: .To detect the influence of reference genes on the outcome of an experiment, the relative expression patterns of two genes SOD and POD were evaluated using the most stable and unstable genes in Days 0, 3, 6 and 9 under drought treatment. […]

Pipeline specifications

Software tools Primer3, BestKeeper, NormFinder, CFX Manager
Application qPCR
Organisms Lolium perenne
Chemicals Abscisic Acid