Technique / Molecular Biology / PCR / RT-PCR

The use of single cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of 5 male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer/probes for beta2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), insulin-like growth factor 1 receptor (IGF1R), and glucose transporter subtype 4 (Glut4). The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used (1/4) of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was less than 8% (CT value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4, PDK4). We observed a 13.9 fold change in expression after resistance exercise which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers. Key words: skeletal muscle, gene expression, Glut4, IGF, PDK4.