Comparison of Three RT-PCR Based Methods for Relative Quantification of mRNA
Davorka Breljak1, Andreja Ambriović-Ristov2, Sanja Kapitanović1, Tamara Čačev1 and Jelka Gabrilovac1
1Ruđer Bošković Institute, Division of Molecular Medicine, Bijenička c. 54, HR–10000 Zagreb, Croatia
2Ruđer Bošković Institute, Division of Molecular Biology, Bijenička c. 54, HR–10000 Zagreb, Croatia
Article history:
Received December 23, 2004
Accepted May 25, 2005
Key words:
aminopeptidase N, APN, HL-60, RT-PCR, real-time PCR, competitive PCR
Summary:
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-γ (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-γ 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.
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