Quantification of Cytokine mRNAs in Human Myocardial Biopsy Samples by Real-Time Quantitative PCR Technology Using the LightCycler Instrument

Understanding the pathophysiology of human heart failure [1–3] necessitates the sensitive measurement of gene expression in human myocardium. Estimating the quantities of mRNAs in an explanted failing human heart sample after heart transplantation is a relatively easy task because of the availability of large-sized tissue samples. However, when the availability of tissue samples is very low, especially from patients who are undergoing therapeutic treatment [4], estimating the quantities of mRNAs requires a highly sensitive method of measurement. In this study, we have developed a highly sensitive method for the quantification of cytokine mRNAs in human myocardial biopsy tissue samples as low as 2–3 mg. This method involves isolation of total RNA from frozen biopsy tissue samples, capturing and immobilizing poly-A RNA, reverse transcription of RNA to obtain single-stranded complementary DNA, and quantification of cytokine mRNAs using a real-time quantitative polymerase chain reaction method.