Real-time RT-PCR has been used to analyze gene expression from single muscle fibers; however, this technique usually involves extracting the RNA first. We analyzed the expression of multiple genes in a procedure in which the single muscle fiber is placed directly into the RT-PCR buffer without any RNA isolation steps. A muscle biopsy was performed on a healthy male subject and the muscle tissue was immediately placed in RNAlater. A small section of the biopsy was then separated, rinsed in pure water, and single muscle fibers were extracted. Fibers were then put in individual tubes containing buffer for the reverse transcription (RT) step of a two-step RT-PCR kit (Invitrogen). After RT, the cDNA was divided into three PCR reactions each containing a separate primer/probe set for GAPDH, beta-2-microglobulin (B2M), and beta-actin. The average (+/− SD) cycle threshold (CT) for the three housekeeping genes was 20.4 ± 1.2, 23.0 ± 1.2, and 28.7 ± 1.6 respectively (n=18). There was no expression in the no-template control and in a minus RT control expression only occurred in B2M at a CT of 32. In addition, we have amplified a common gene of interest, insulin-like growth factor 1 receptor (IGF1), along with B2M. The average cycle threshold for B2M was 23.6 ± 1.1 and IGF1 was 29.6 ± 1.0 (n=25). These results show that consistent gene expression results can be obtained from this procedure. (Tehel and McCabe were supported by NIGMS R25 GM62232)