Computational protocol: Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm

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Protocol publication

[…] To isolate the most stable and reliable reference genes in the process of endosperm development under different temperature conditions, specific primers for the above 8 candidate reference genes were designed using the Primer 3 ( The specific primers of housekeeping genes, Actin eEF-1a, GAPDH, β-TUB, eIF-4a, UBQ10, UBC, UBQ5, 17S rRNA, 18S rRNA and 25S rRNA were amplified as described previously [].To check specificity of these primers, qPCR was performed using the iQ™ SYBR® Green PCR Super Mix (Bio-Rad) on CFX96 Real-Time PCR Detection System and the PCR products were analyzed by melting curves and also on 2% ethidium bromide stained agarose gel. Each qRT-PCR reaction mix containing 0.5 μl of 1: 5 diluted cDNA, 4 μl DNase/RNase free water H2O, 5 μl 2× iQ™ SYBR® Green PCR Super Mix, and 0.5 μl of the forward and reverse primers. The following amplification program was used: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation for 10 s at 95°C, annealing for 10 s at 58°C, and extension for 10 s at 72°C. Melting curve data were collected from 65°C to 95°C in 0.5°C increments. The real-time PCRs were performed on CFX96 Real-Time PCR System (Bio-Rad), the threshold Cycle (Ct) was auto-calculated by the CFX96 Manager Software (Bio-Rad). [...] Two widely used algorithms, NormFinder and geNorm were used to analyze the stability of the candidate reference genes under different experimental conditions. Ct values of the candidate reference genes were imported into software according to the corresponding manuals of NormFinder [] and geNorm v3.5 [] algorithms, respectively, to evaluate gene expression stability.The expression stability value (M) for all genes and mean pairwise variation of a gene from all other reference genes in a given set of samples were calculated by the geNorm algorithm. All the candidate reference genes are ranked based on their stability in a given set of samples, and the number of reference genes necessary for an optimal normalization is indicated as well.The gene expression stability with optimal normalization among a set of candidate reference genes were analyzed by NormFinder algorithm. The NormFinder ranks the stability of candidate reference genes and calculates not only the candidate reference gene variation but also the variation between sample subgroups of the sample set. The lowest stability value indicates the most stable candidate reference genes within the gene set examined. […]

Pipeline specifications

Software tools Primer3, NormFinder
Application qPCR
Organisms Oryza sativa