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

Davorka Breljak1, Andreja Ambriović-Ristov2, Sanja Kapitanović1, Tamara Čačev1 and Jelka Gabrilovac1


1
Ruđer 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.

 


*Corresponding author:    breljak@irb.hr

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