Optimization of Real Time PCR for Precise Measurement of HER۲ Overexpression in Breast Cancer Specimens

Background: Breast cancer is one of the most prevalent malignancies among women in various countries. HER۲ overexpression, which is due to different reasons, occurs in ۲۰-۳۰% of breast cancers. HER۲ gene encodes an ۱۸۵kDa transmembrane glycoprotein with ۱۲۵۵ amino acids. This active product triggers downstream intracellular signaling pathways inducing cell proliferation and cell survival. These activities can be done in an uncontrolled manner in the cases which HER۲ expression undergoes up-regulation. The aim of this study was optimization of Real Time PCR condition. Materials and Methods: RNA purification, cDNA synthesis and then optimization of Real Time PCR method performed respectively. In this study, total RNA was extracted from fresh tissue samples, first strand of total cDNA was synthesized and in the following steps, Real Time PCR was performed to be optimized. Results: Although altering the protocol, annealing temperature and concentration of MgCl۲ did not make any improvement and beneficial effects on reactions, changing the concentration of primers to ۰.۲۴ pm/;mul was influential to eliminate primer dimers of Real Time PCR reactions. It demonstrated that the copy number of GAPDH transcripts is more than HER۲ transcripts in normal breast tissues. Therefore, deviation in ۲.۵ differences between the Ct value of HER۲ and GAPDH indicated that the copy number of HER۲ transcripts was increased therefore, HER۲ underwent overexpression in these cases. Conclusion: Under these optimized conditions, this technique can be applied as a powerful method in clinical laboratories.

کلیدواژه ها:

HER۲ gene ، total cDNA ، RNA purification ، Real Time PCR