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Title: The Use of Comparative Quantitative RT-PCR to Investigate the Effect of Cysteine Incubation on GPx1 Expression in Freshly Isolated Cardiomyocytes
Contributor(s): King, Nicola  (author)
Publication Date: 2010
DOI: 10.1007/978-1-60761-629-0_14
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Abstract: 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°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.
Publication Type: Book Chapter
Source of Publication: RT-PCR Protocols, p. 215-232
Publisher: Springer
Place of Publication: New York, United States of America
ISBN: 1607616289
Field of Research (FOR): 110201 Cardiology (incl Cardiovascular Diseases)
060107 Enzymes
060602 Animal Physiology - Cell
HERDC Category Description: B1 Chapter in a Scholarly Book
Series Name: Methods in Molecular Biology
Series Number : 630
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Appears in Collections:Book Chapter
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