Computational protocol: Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean

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Protocol publication

[…] Primers from published literature with good specificity and amplification efficiency were utilized in our study (). All primers were synthesized by Invitrogen (Shanghai, China). Quantitative RT-PCR experiments were carried out using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Japan) on LightCycler 480 (Roche, Switzerland). The program of the qRT-PCR was 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 15 s and 72°C for 15 s. Dissociation curves were obtained using a thermal melting profile performed after the last PCR cycle: 95°C decreases to 40°C at the speed of 5°C per second followed by a constant increase in the temperature between 60°C and 95°C. The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines proposed the use of quantification cycle (Cq) value over the threshold cycle (Ct) according to the RDML (Real-Time PCR Data Markup Language) data standard []. Cq value represents the number of cycles when the density of fluorescence meets the set threshold. Therefore, Cq values were used in this study. Each sample was tested in three technical replicates.A series of 10-fold dilutions of cDNA templates (10–1,000 fold dilution) were made to generate standard curves, and the gene specific amplification efficiency for each primer pair in qRT-PCR was determined by the slope of the log-linear portion of the calibration curve. The gene specific PCR amplification efficiency (E) is calculated by using the equation: E (%) = (10−1/slope-1) × 100% []. The relative expression levels of target genes were calculated using the 2-ΔΔCT method []. [...] The stabilities of reference genes were analyzed using software tools, including geNorm [], BestKeeper [], NormFinder [], Delta Ct [], and RefFinder [], following the corresponding instructions. The geNorm program identifies the most stable reference genes based on the average pairwise variation of a reference gene with other housekeeping genes, and ranks the reference genes by their expression stability values (M). In general, the lower the M value, the higher is the expression stability of candidate genes []. BestKeeper determines the “optimal” reference genes on the basis of pair-wise correlation analysis of all pairs of candidate reference genes []. NormFinder calculates the overall variation of the candidate reference genes in all samples and also the variation of intra- and inter-groups []. Delta Ct compares the relative expression of pairs of candidate genes within each sample. If the ΔCt value between two reference genes does not change among different samples, it indicates either both genes have stable expression patterns or they are co-regulated among the samples, yet the different ΔCt value suggests that at least one of them is variably expressed. Then the third, fourth, or more genes are introduced into the comparisons to find out which pairs show less variability, and hence which gene has stable expression among the samples tested. Ultimately, an appropriate reference gene can be selected for a particular experimental system []. Finally, RefFinder generates a comprehensive ranking by calculating the geometric mean of each reference gene in the above four methods, in which the smaller the ranking, the more stable is the reference gene [–]. […]

Pipeline specifications

Software tools RDML, BestKeeper, NormFinder
Application qPCR
Organisms Glycine max
Chemicals Aluminum, Cadmium