Reverse transcription and real-time PCR have become the method of choice for the detection of low-abundance mRNA transcripts obtained from small human muscle biopsy samples. GAPDH, beta-actin, beta-2M, and 18S rRNA are widely employed as endogenous control genes, with the assumption that their expression is unregulated and constant for given experimental conditions. The aim of this study was to determine if mRNA transcripts could be performed on isolated human single muscle fibers and to determine reliable housekeeping genes (HKGs) using quantitative gene expression protocols at rest and in response to an acute exercise bout. Muscle biopsies were obtained from the gastrocnemius of three adult males before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO(2)max. A total of 40 single fibers (MHC I and IIa) were examined for GAPDH, beta-actin, beta-2M, and 18S rRNA using quantitative RT-PCR and SYBR Green detection. All analyzed single fiber segments showed ribosomal RNA (28S/18S). No degradation or additional bands below ribosomal were detected (rRNA ratio 1.5-1.8). Also, no high or low-molecular weight genomic DNA contamination was observed. For each housekeeping gene the duplicate average SD was +/-0.13 with a CV of 0.58%. Stable expression of GAPDH was observed at all time points for each fiber type (MHC I and IIa). Inconsistent expression of beta-actin, beta-2M, and 18S rRNA was observed during the post-exercise time points for each fiber type. These data indicate that successful extraction of high quality RNA from human single muscle fibers along with quantification of mRNA of selected genes can be performed. Furthermore, exercise does influence the expression of certain HKGs with GAPDH being the most stable. (C) 2004 Elsevier Inc. All rights reserved.