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. 2019 Oct 2;9(1):14147.
doi: 10.1038/s41598-019-50667-2.

Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents

Benshui Shu  1   2   3 , Jingjing Zhang  2   3 , Jie Zeng  2   3 , Gaofeng Cui  2   3 , Guohua Zhong  4   5
Affiliations

Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents

Benshui Shu et al. Sci Rep. .

Abstract

As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms. Previous study have demonstrated that apoptosis occurred in Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. However, the selection of reference genes influences the accuracy of results profoundly. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin; RPL13 and RPL3 for cells treated with camptothecin; EF1α and β-1-TUB for cells irradiated under ultraviolet; and EF1α and EF2 for combinational analyses of samples. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Melting curves of eight candidate reference genes. Eight candidate reference genes were showed as Actin, EF1α, EF2, GAPDH, RPL3, RPL13, α-TUB and β-1-TUB.
Figure 2
Figure 2
Expression profiles of eight candidate reference genes in Sf9 cells under different conditions.
Figure 3
Figure 3
Comprehensive ranking of eight candidate reference genes in Sf9 cells with different treatments analyzed by RefFinder. The reference gene with more stable expression has the lower Geomean of ranking value.
Figure 4
Figure 4
The Pairwise variation values in different experimental conditions according to geNorm. The value of Vn/Vn + 1 < 0.15 means n should be the optimal number of reference gene selection in RT-qPCR analysis.
See this image and copyright information in PMC

References

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