RT-PCR Protocols

King N, Nicola King, King, Nicola

Intracellular cysteine availability is one of the major rate limiting factors that regulate the synthesis of the major antioxidant, glutathione. Little is known, however, about the effect of cysteine upon glutathione-associated enzymes in isolated heart cells. Such knowledge is important if a full understanding and exploitation of cysteine's cardioprotective potential is to be achieved. Therefore, this study describes the use of a comparative quantitative reverse transcription polymerase chain reaction (RT-PCR) assay to investigate the effect of incubation of freshly isolated rat cardiomyocytes for 2 h at 37 degrees C with or without 0.5 mM cysteine on the expression of cellular glutathione peroxidase (GPx1). The main analytical method is the conventional RT-PCR in a standard thermal cycler followed by electrophoresis and scanning densitometry using the expression of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), for normalising purposes. Each step of this straight-forward and relatively inexpensive method is explained in detail to facilitate its adoption by the reader for experiments investigating the effects of any compound on any gene in any cell population. The results of the current investigation show that cysteine incubation significantly increases the expression of GPx1 in freshly isolated cardiomyocytes compared to control, suggesting the possibility of a new beneficial role for cysteine in myocardial protection.