Comparison of Three RT-PCR Based Methods for Relative Quantification of mRNA

• Main Authors: Andreja Ambriović-Ristov, Sanja Kapitanović, Tamara Čačev, Jelka Gabrilovac, Davorka Breljak
• Format: Article
• Language: English
• Published: University of Zagreb 2005-01-01
• Series: Food Technology and Biotechnology
• Subjects: aminopeptidase N; APN; HL-60; RT-PCR; real-time PCR; competitive PCR
• Online Access: http://hrcak.srce.hr/file/162952

Comparison of three RT-PCR based methods: semi-quantitative, competitive and real--time RT-PCR for relative quantification of mRNA is presented. Aminopeptidase N expressed on human promyeloid HL-60 cell line, at basal and activated state, served as a model for comparison. HL-60 cells were stimulated with IFN- (6 ng/mL) for 72 h at 37 oC, total cellular RNA was isolated, reverse transcribed to cDNA and semi-quantitative, competitive and real-time RT-PCR were performed to obtain the relative levels of mRNA for aminopeptidase N. The data obtained showed that all three RT-PCR based methods gave reliable and comparable results, i.e. approximately twofold increase of aminopeptidase N mRNA on IFN- stimulated HL-60 cells. Thus, in spite of rapid advances made in the area of real-time RT-PCR, end-point RT-PCR such as competitive and semi-quantitative RT- -PCR, although laborious and time consuming, may still remain useful techniques for relative mRNA quantification when small number of samples are to be analyzed.