JIA-2018-09

2058 ZHANG Bai-zhong et al. Journal of Integrative Agriculture 2018, 17(9): 2054–2065 stability rankings from the most stable to the least stable were as follows: α-TUB , 28S , 18S , TBP , EF1β , ACT , GAPDH , and RPL18 (Fig. 2-A). In addition, RPL18 was identified by all the four methods as the least stably expressed reference gene. Moreover, using geNorm analysis, the V 2/3 was below the threshold of V=0.15 (Fig. 3), indicating that two most stable genes are required for normalization. Therefore, for the developmental stage experiments, α-TUB and 28S were appropriate to normalization. Different tissues ∆Ct method and NormFinder identified 18S and ACT as the most stably expressed genes, BestKeeper selected 28S and α-TUB , while geNorm selected EF1β and TBP (Table 2). According to the RefFinder method, the stability rankings from the most stable to the least stable were as follows: 18S , ACT , α-TUB , 28S , EF1β , TBP , RPL18 , and GAPDH (Fig. 2-B). Interestingly, GAPDH was identified by four methods as the least stably expressed reference gene. For geNorm analysis, all of Candidate reference genes Cycle threshold values (Ct) Ef1β TBP α-TUB 18S 28S GAPDH ACT RPL18 20 25 30 35 Fig. 1 Expression levels of candidate reference genes of Schizaphis graminumas indicated by Ct values. A higher Ct value indicates lower level of expression. The black boxes indicate the mean values of all samples ( n =27), and the whiskers indicate the standard error of the mean. Table 2 Expression stability of the candidate reference genes under different experimental conditions Condition Reference gene ∆Ct BestKeeper NormFinder geNorm Stability Rank Stability Rank Stability Rank Stability Rank Developmental stage EF1β 1.765 5 1.103 6 1.410 6 0.569 4 TBP 1.718 4 0.936 5 1.149 4 0.683 5 α-TUB 1.473 2 0.510 1 0.450 1 0.321 1 18S 1.566 3 0.756 3 1.028 3 0.441 3 28S 1.452 1 0.515 2 0.582 2 0.329 2 GAPDH 2.704 7 1.944 7 2.311 7 1.572 7 ACT 2.076 6 0.843 4 1.341 5 1.022 6 RPL18 2.293 8 2.268 8 3.144 8 2.002 8 Different tissues EF1β 0.793 5 1.080 7 0.615 5 0.231 1 TBP 0.828 7 1.016 6 0.696 6 0.236 2 α-TUB 0.724 3 0.657 2 0.407 3 0.413 4 18S 0.681 1 0.917 4 0.342 2 0.279 3 28S 0.764 4 0.527 1 0.442 4 0.492 5 GAPDH 1.304 8 1.243 8 1.222 8 0.855 8 ACT 0.713 2 0.823 3 0.160 1 0.557 6 RPL18 1.032 7 0.966 5 0.847 7 0.705 7 Insecticide treatment EF1β 1.718 6 1.529 8 1.476 6 0.707 5 TBP 1.338 3 1.110 4 0.651 3 0.391 3 α-TUB 1.323 1 1.037 3 0.599 2 0.270 1 18S 1.603 4 1.407 6 1.294 5 0.621 4 28S 1.335 2 0.875 2 0.495 1 0.273 2 GAPDH 1.686 5 0.498 1 0.923 4 1.080 6 ACT 2.513 8 1.442 7 2.271 8 1.746 8 RPL18 2.452 7 1.152 5 2.189 7 1.490 7 Pooled sample EF1β 1.672 5 1.561 6 1.305 5 0.645 4 TBP 1.537 4 1.318 4 0.879 3 0.690 5 α-TUB 1.431 2 1.205 3 0.587 2 0.362 1 18S 1.513 3 1.454 5 0.996 4 0.568 3 28S 1.401 1 1.082 2 0.485 1 0.369 2 GAPDH 2.282 6 1.714 7 1.819 6 1.587 7 ACT 2.453 7 1.032 1 2.037 7 1.243 6 RPL18 2.769 8 2.309 8 2.532 8 1.882 8