Computational protocol: Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus )

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Protocol publication

[…] Thirteen reference genes were selected for investigation to identify the most stably expressed reference gene(s) to be used in RT-qPCR studies. Reference genes were chosen based on literature and nucleotide sequences were recovered from the Ensembl database (www.ensembl.org/). These sequences were used to design primers by the PrimerQuest software provided by Integrated DNA Technologies, Inc (IDT, Coralville, IA, USA). Additionally, BLAST analyses were performed to verify their specificity. The primers sequence information used on this study is shown in and the primer specificity in the .Prior to the RT-qPCR amplification, part of cDNA was pooled into a new tube to make one composed sample which was then diluted to construct the standard curves for PCR conditions optimization and calculation of PCR efficiency. For this purpose, four cDNA quantity (1, 5, 15, 45 ng) and four primers concentrations (200, 400, 800 and 1000 nM) were tested. The following experimental RT-qPCR conditions were used: 1 cycle of 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60 sec at 60–62°C. Additional steps with a gradual increase in temperature from 60–62 to 95°C were used to obtain the dissociation curve. After the analysis of efficiency, the most adequate annealing temperature and primer concentration was used to perform PCR reaction. A reaction mix without template was also used to detected possible reagent contamination. The PCR amplification reaction was performed at different wells and in duplicates. Each primer pair was checked for size specificity of the amplicon by 1.5% agarose gel electrophoresis and ethidium bromide staining. The PCR amplification efficiencies were calculated for each target and reference gene assay using the formula E = (10-1/slope—1) × 100 []. [...] To prepare data set input, Cq values were transformed to relative quantities using the formula (1+E)-∆Cq []. The sample with Cq maximum expression was used as the calibrator with a set value of 1 ((1+E)-Cqoriginal—Cqmax). Thus, the lowest value was zero. To calculate the stability of the candidate genes we have used the BestKeeper [], NormFinder [], geNorm Excel [], geNorm SAS [] and ΔCt method [] were used. Relative quantities was used as the input to perform the analysis on NormFinder, geNorm and ΔCt method. No transformed Cq values are required for BestKeeper analysis. In addition, geNorm was used to determine the optimal number and the reference genes required for reliable normalization of RT-qPCR data. All data were analyzed according to the instructions for each software tested. […]

Pipeline specifications

Software tools PrimerQuest, BestKeeper, NormFinder
Application qPCR
Organisms Gallus gallus
Chemicals Carbon Dioxide