Optimization of Real Time PCR method for precise measurement of HER2 overexpression in breast cancer specimens

Breast cancer is one of the most prevalent malignancies among women in various countries. HER2 overexpression, which is due to different reasons, occurs in 20-30% of breast cancers. HER2 gene encodes an 185kDa transmembrane glycoprotein with 1255 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 HER2 expression undergoes up-regulation. The aim of this study was optimization of Real Time PCR condition. 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. Although altering the protocol, annealing temperature and concentration of MgCl2 did not make any improvement and beneficial effects on reactions, changing the concentration of primers to 0.24 pm/μl was influential to eliminate primer dimers of Real Time PCR reactions. It demonstrated that the copy number of GAPDH transcripts is more than HER2 transcripts in normal breast tissues. Therefore, deviation in 2.5 differences between the Ct value of HER2 and GAPDH indicated that the copy number of HER2 transcripts was increased; therefore, HER2 underwent overexpression in these cases. Under these optimized conditions, this technique can be applied as a powerful method in clinical laboratories.