Identification of Housekeeping Genes Suitable for Gene Expression Analysis in Rat Breast Cancer Cell Line Treated with Doxorubicin and Piperine

Background Real-time quantitative PCR (qRT-PCR) is often used as an effective experimental method for analyzing gene expression. In this method, normalization of target gene expression levels must be performed using housekeeping genes (HKGs). HKGs are used to compensate for difference between samples due to diverse quality and quality of RNAs and different reverse transcription yield. For an ideal reference gene, constant expression levels across different samples of one experiment is necessary. In the current study, expression stability of four candidate references genes including Beta actin (ACTB), glyceraldeyde-۳- phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT۱) and ribosomal protein L۱۳a (RPL۱۳A) following doxorubicin-piperine treatment in rat breast cancer cell line were evaluated. Methods LA۷ cell line was exposed to doxorubicin-piperine for ۵ days. RT-qPCR for candidate references genes was performed and normalization between untreated and doxorubicin-piperine - treated cells was performed using identical sample input amounts. Results RT-qPCR results indicated significant difference in expression level of GAPDH, ACTB and HPRT between untreated and treated cells. However, the transcriptional level of RPL۱۳A remained unchanged after treatment. Conclusion RPL۱۳A was recognized as valid reference genes for analysis of gene expression during doxorubicin-piperine treatment of LA۷ cells, while GAPDH, ACTB and HPRT were not suitable.

Keywords:

Housekeeping genes , Rat breast cancer cell , Doxorubicin , Piperine , expression analysis