Computational protocol: Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity

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[…] The RefGenes tool from Genevestigator was used in order to identify new candidate reference genes []. Due to the absence of experiments with stress by iron excess, conditions of iron deficiency were used in the search of these genes. Genes with low variation in Genevestigator and confirmed by our group as less variable were chosen [].The RT-qPCR followed the guidelines of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) []. The design of primers for RT-qPCR analysis was performed using the Applied Biosystems Primer Express® program according to the following parameters: annealing temperature of 60–65°C, GC content of 40–60% and amplicon size of 50–150 bps. All primer sequences and relevant information are presented in . Dissociation curves were evaluated and only primers giving single peaks were used.Each reaction was performed in triplicate in an ABI 7500 Fast Real-Time PCR System using SYBR R Green I (Life Technologies®, Carlsbad, CA, USA). Each PCR reaction mix consisted of 2 μL of SYBR Green (1:10000), 0.4 μL of forward and reverse oligos (10 mM), 2 μL of PCR buffer (10x), 0.05 μL of dNTPs (10 mM), 1.2 μL of MgCl2 (50 mM) and 0.05 μL of Platinum R Taq DNA Polymerase (Life Technologies®, Carlsbad, CA, USA; 2 U/rxn) in a total volume of 10 μL. Finally, 10 μL of 1:50 diluted template cDNA was added, resulting in a total volume of 20 μL per PCR reaction. PCR cycling was performed as follows: 5 min at 94°C followed by 40 rounds of 15 s at 94°C, 10 s at 60°C, 15 s at 72°C, and finally 1 round of 35 s at 60°C. Melting curve cycling consisted of: 15 s at 95°C, 1 min at 60°C, 30 s at 95°C, and 15 s at 60°C. [...] Real-time PCR Miner [] was used to evaluate primer efficiency. The efficiency (E) of each primer pair was greater than 90% in all experimental sets, indicating that the amount of PCR product nearly doubled after each cycle. The Ct values were submitted, trough RefFinder [] (, to four different methods that rank the best constitutive genes, geNorm [], NormFinder [], BestKeeper [] and the comparative ΔCt method []. The softwares are based on different algorithms using information as Ct analysis of the genes in different tissues and groups of three biological replicates and initiator efficiency in each biological sample. This analysis identified genes that show the smallest variation in the number of transcripts in each tissue. The relative expression data were calculated according to the 2-ΔΔCt method []. […]

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