Reliability of RT-PCR methods for measuring relative gene expression in mast cells

• Published in: Veterinary immunology and immunopathology Vol. 100; no. 1; pp. 1 - 5
• Main Authors: Ikeda, Teruo, Murakami, Masaru, Funaba, Masayuki
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2004
• Subjects: Animals; Gene Expression Regulation - physiology; Gene transcripts; Linear Models; Mast cells; Mast Cells - physiology; Mice; PCR; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Mast cells; PCR; Gene transcripts
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/j.vetimm.2004.02.011

Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels. Results from competitive RT-PCR analysis correlated closely with those from real-time RT-PCR analysis. When the differences in mRNA level between samples are relatively smaller, however, the correlation tended to be weaker. Real-time RT-PCR analysis has higher reliability, but is expensive. In contrast, competitive RT-PCR analysis is inexpensive, but is weaker at detecting smaller differences in gene transcript level between samples. Therefore, the most appropriate analytical method to measure mRNA levels should be chosen, depending on the experimental conditions.