Comparative quantification of target gene expression in Breast cancer cells byReal-time PCRmethod

The purpose of this research was to develop a quantitative real-time PCR for CK19 (Cytokeratin 19) - mRNA and evaluate its potential for the molecular detection of breast cancer cell lines.Methods: The method is based on real-time PCR (Polymerase Chain Reaction) by fluorescent SYBR green. RNA was prepared from MCF7, MDA-MB-231, SKBR3 and T47D breast carcinoma cells and cervical cancer cell line (HeLa) as a negative control. This RNA was screened for mRNA of CK19and Glyceraldehyde 3-phosphate Dehyrogenase (GAPDH) by real-time PCR and the results validated by Electrophoresis. The average ΔCt of CK19 and GAPDH was calculated in each cell line as well as the ΔCtof GAPDH from different cell lines. Quantitative CK19 expression was calculated using the ΔΔCt method.Results: Variable degrees of expression of CK19 was detected in MCF7, T47D and SKBR3 cell lines while MDA-MB-231 cell line was not expressed CK19 by this method. Conclusions: Real-time PCR technique is sensitive, accurate and reliable method which can be used for high throughput comparative quantification oftrnnor marker studies

Keywords:

Breast cancer , cell line , CK19 , Real-time PCR