Computational protocol: Validation of Reference Genes Aiming Accurate Normalization of qRT-PCR Data in Dendrocalamus latiflorus Munro

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Protocol publication

[…] Twelve reference genes except TIP41 and NTB and two target genes were selected from the transcriptome of D. latiflorus Munro and gene sequences were deposited in the GenBank (accession numbers are listed in ). The coding sequences for TIP41 and NTB were first amplified by homologous cloning on the basis of the sequence information from Moso bamboo, then sequenced, characterized and submitted to the Genbank. The primers for the twelve reference genes and two target genes were designed using Primer 3 software ( (). In order to confirm the primer specificity, all primer pairs were initially tested via standard RT-PCR using the Premix Ex Taq (TaKaRa, Japan) and a single amplification product of the expected size for each gene was verified by electrophoresis on a 2% agarose gel and stained with ethidium bromide. Negative PCR control without templates was also conducted for each primer pair. qRT-PCR reactions were performed in 96-well plates with an Applied Biosystems 7300 Real-Time PCR system using SYBR® Premix Ex Taq™ (TaKaRa, Japan) in a 20 µl reaction volume (containing 2 µl cDNA reaction mixture, 10 µl SYBR® Premix Ex Taq™, 0.4 µl ROX Reference Dye, and 0.4 µl each primer). The thermal profile of the reaction followed the instructions that recommended by the manufacturer (10 s at 95oC, 40 cycles of 95oC for 5 s, and 60oC for 31 s). Melting dissociation curve, obtained by heating the amplicon from 60oC to 95oC, was carried out to verify the presence of gene-specific peaks and the absence of primer dimers. All qRT-PCR reactions were performed in technical triplicate. The final threshold cycle (Ct) values were the mean of four values. [...] The slope of a standard curve serves as an indication of the efficiency of the qRT- PCR. To generate a standard curve, Ct values of 10-fold series dilution of the mixed cDNA template for each primer pair are plotted against the logarithm of input amount of standard material. The correlation coefficients (R2) and the slope could be calculated from the resulting standard curve and amplification efficiencies (E) could be determined from the given slope generated in Microsoft Excel 2010 according to the equation E = (10−1/slope−1)×100 .In order to identify a suitable reference gene, the gene expression stability was statistically analyzed based on the results of three different types of Microsoft Excel-based software: geNorm , NormFinder , and BestKeeper . GeNorm is an algorithm that selects an ideal pair of reference genes out of candidate genes and it relies on the principle that the expression ratio of two ideal reference genes should not be influenced by external factors, regardless of the experiment condition or tissue type. Through the calculation and comparison of the reference gene expression stability measure (M value) of all candidate genes, the gene with highest M-value was eliminated and the process was repeated until only two genes left. Genes with low M values were characterized as the most stable expression gene.The NormFinder reference tool, another algorithm that attempts to find the optimum reference genes out of a group of candidate reference genes, could calculate the variance within group as well as the variance between groups. According to the analysis, the candidate reference gene expression stability will be estimated and the gene with the lowest stability value will be top ranked.BestKeeper evaluates the stabilities of candidate reference genes based on the coefficient of correlation to the BestKeeper index, which is the geometric mean of the Ct values of all candidate reference genes , . The coefficient of variance (CV) and the standard deviation (SD) of the Ct values are also calculated using the whole data set and all the Ct values are analyzed as a total data set . Reference genes exhibiting the lowest coefficient of variance and standard deviation (CV±SD) are identified as the most stable genes. Genes showing a SD greater than 1 are considered to be unacceptable .The above three software packages were used following the manufacturer’s protocols. For geNorm and NormFinder, the raw Ct values needed to be transformed into the required data input format. The maximum expression level of each gene indicated by the lowest Ct value was used as a control and was set to a value of 1. Relative expression levels were then calculated from Ct values using the formula: 2−ΔCt, in which ΔCt = each corresponding Ct value–minimum Ct value. The obtained results were then further analyzed with geNorm and NormFinder. BestKeeper analyses were based on untransformed Ct values. […]

Pipeline specifications

Software tools Primer3, NormFinder, BestKeeper
Application qPCR
Organisms Dendrocalamus latiflorus