Introduction: Hypoxia-mediated tumor metastasis, progression and drug resistance are major clinical challenges in ovarian cancer. Meanwhile, the genetic basis of these traits is still not clear. RT-qPCR, as an efficient and sensitive gene expression technique, has been widely used for gene analyses, providing a basis for in-depth understanding of molecular changes in different microenvironments. However, there is currently a lack of suitable reference genes to normalize the data associated with hypoxia in ovarian cancer cells.
Methods: A systematic method is needed to select the most suitable reference gene. Here, eight candidate reference genes (GAPDH, β-actin, 18S RNA, TUBB, PPIA, TBP, RPL13A and SDHA) from humans were selected to assess their expression levels in SKOV3 cells under hypoxia. The geNorm and NormFinder programs were utilized to evaluate the expression stabilities of these selected candidate reference genes.
Results: Interestingly, 18S RNA was considered to be an ideal reference gene for the normalization of target gene expression under hypoxic conditions. Furthermore, this result was confirmed in another two ovarian cancer cell line, CAOV3 and OVCAR3 cell line. Finally, these results suggest that appropriate reference genes should be selected before performing gene expression analysis during hypoxic environmental exposure.
Conclusion: 18S RNA can be used as an appropriate reference gene for the study of gene expression in ovarian cancer samples under hypoxia by RT-qPCR.
Keywords: reference genes, ovarian cancer, hypoxia, EMT