JIA-2018-09

2057 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 the cycle threshold (Ct) value vs . the log of the serially diluted template concentration. The optimised RT-qPCR program consisted of an initial step at 50°C for 2 min, 94°C or 2 min, followed by 50 cycles of 94°C for 15 s and 60°C for 30 s. After the cycling protocol, melting curves were obtained by increasing the temperature from 60 to 95°C (0.2°C s −1 ) to denature the double-stranded DNA. The RT-qPCR amplifications were carried out in 96-well plates. The assays were run in an ABI 7500 System using the SDS ver.1.4 application software (Applied Biosystems, USA). Standard curves were created based on a five-fold dilution series of cDNA (1:5, 1:25, 1:125, 1:625, 1:3 125, and 1:15 625). Each sample was prepared as three biological replicates, and each reaction was analyzed with two technical replications. 2.7. Analyses of the stability of reference gene ex- pression The geNorm software initially calculates the value of gene expression stability (M) and generates a stability ranking; genes with the lowest M value have the most stable expression. The geNorm also calculates pair-wise variation V n / n +1 , which represents the variation between two sequential normalization factors and determines the optimal number of reference genes required for accurate normalization. A V n /V n +1 ratio below 0.15 suggest that the use of an additional reference gene would not significantly improve normalization. NormFinder software is a model- based approach to identify suitable reference genes for use in normalization (Andersen et al. 2004), the candidate gene with the lowest value is considered the most stable reference gene. Excel-based software BestKeeper and the comparative ∆Ct method were also used to select optimal reference genes. A user-friendly web-based comprehensive tool, RefFinder online (http://150.216.56.64/ referencegene.php) was used to evaluate and select reference genes. RefFinder combines the aforementioned major computational programs (∆Ct method, geNorm, Normfinder, and BestKeeper) to compare and rank the tested candidate reference genes. RefFinder also assigns an appropriate weight to each gene and calculates the geometric mean of the weights for the final ranking. 2.8. Validation of reference gene selection To evaluate the validity of the selection of reference genes, the expression levels of three target genes, HSP70 , SgraCYP18A1 , and GST were analyzed under different experimental conditions (different developmental stages, tissues, and insecticide treatments). For each experimental condition, the expression profiles of HSP70 , SgraCYP18A1 , and GST were normalized using the most stable reference gene (NF1), the least stable reference gene (NF8) and several stable reference genes (NF(1 –n )) recommended by RefFinder. The relative expression levels of HSP70 , SgraCYP18A1 , and GST in different samples were calculated according to the 2 –∆∆C T method (Livak and Schmittgen 2001). 2.9. Statistical analysis Data organization was performed using Excel 2010 and lethal concentration of pesticides was calculated using Polo (probit and logit analysis) (LeOra Software Company, Petaluma, CA). The expression levels of target genes one- way ANOVA using InStat v.3.0 (GraphPad Software, San Diego, CA) with a significance level set at P <0.05. 3. Results 3.1. Traditional PCR amplification efficiencies and expression levels of candidate reference genes Traditional PCR was used to evaluate the primer specificity of the eight reference genes and the three target genes of interest. Each primer pairs produced a single product through PCR, then analysis of melting curves showed that there was a single peak for each primer pairs. The amplification efficiencies of all the primer pairs were between 91.3 and 108.2%, and the coefficient of determination ( R 2 ) ranged from 0.990 to 0.999 (Table 1). The Ct values were adopted to compare the transcript abundance of the selected genes in different samples. The mean Ct values of the eight reference genes varied significantly. The means of the Ct values ranged from 21.05 to 27.60, with the lowest and highest Ct values obtained from EF1β (21.05) and ACT (27.60), respectively. EF1β had the highest mean expression levels, followed by 18S (23.73), 28S (24.13), α-TUB (24.23), TBP (25.72), GAPDH (26.05) and RPL18 (27.41) (Fig. 1). 3.2. Stability of the candidate reference gene ex- pression Analyses using four programs ranked the tested genes according to gene stability measured from the most stable (the lowest value) to the least stable (the highest value) as shown in Table 2. Different developmental stages The overall expression stability rankings produced by the four methods, ∆Ct method, BestKeeper, NormFinder, and geNorm, were almost identical, the top two stable reference genes were α-TUB and 28S (Table 2). According to the RefFinder method, the