JIA-2018-09

2059 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 the V n/n +1 values were below the 0.15 cut-off value (Fig. 3), indicating that the two most stable genes are required for normalization. Therefore, for the different tissues, 18S and ACT were appropriate to normalization. Insecticide treatments The stability rankings produced by ∆Ct, NormFinder, and geNorm were similar, in that α-TUB and 28S were confirmed as the two most stably expressed reference genes, and ACT was identified by all the these three methods as the least stably expressed reference gene. BestKeeper identified GAPDH and 28S as the most stably expressed reference genes (Table 2). According to the RefFinder method, the stability rankings from the most stable to the least stable were as follows: 28S , α-TUB , TBP , GAPDH , 18S , EF1β , RPL18 , and ACT (Fig. 2-C). For geNorm analysis, the V 2/3 was below the 0.15 cut-off value (Fig. 3). Thus, two reference genes were enough to normalize the gene expression levels in RT-qPCR analyses. Therefore, 28S and α-TUB were the most suitable for normalizing RT-qPCR data in the insecticide treatments. Pooled data of various conditions Stability rankings produced by ∆Ct method, NormFinder, and geNorm were similar, 28S and α-TUB were confirmed as the two most stably expressed reference genes. However, BestKeeper identifed ACT and 28S as the two most stably expressed reference genes (Table 2). According to the RefFinder method, the stability rankings from the most stable to the least stable were as follows: 28S , α-TUB , 18S , TBP , ACT , EF1β , GAPDH , and RPL18 (Fig. 2-D). Interestingly, RPL18 was identified by all the five methods as the least stably expressed reference gene. For geNorm analysis, the V 2/3 was below the 0.15 cut-off value (Fig. 3). 28S and α-TUB were suggested suitable for normalizing the RT-qPCR data in the insecticide treatments. 3.3. Validation of reference gene selection To assess the stability of selected reference genes, the expression levels of HSP70 , SgraCYP18A1 , and GST were analyzed in the same experimental conditions used for the comparisons of the expression stability of the reference genes. The similar expression levels were obtained in the developmental stages when normalized using the most stable reference gene ( α-TUB ), the combination of the two most stable reference genes ( α-TUB and 28S ), and the 0 2 4 6 8 10 A B C D Candidate reference genes Geomean of ranking values α-TUB 28S 18S TBP EF1β ACT GAPDH RPL18 Geomean of ranking values 18S ACT α-TUB 28S EF1β TBP RPL18 GAPDH 0 2 4 6 8 10 Candidate reference genes Candidate reference genes Geomean of ranking values 28S α-TUB 18S TBP ACT EF1β GAPDH RPL18 0 2 4 6 8 10 Candidate reference genes Geomean of ranking values 28S α-TUB 18S TBP ACT EF1β GAPDH RPL18 0 2 4 6 8 10 Fig. 2 Expression stability of the candidate reference genes under different experimental conditions calculated by RerFinder. A, different developmental stages. B, different tissues. C, insecticide treatments. D, pooled samples. A lower Geomean of ranking value indicates more stable expression.