title: Laser capture microdissection of metachromatically stained skeletal muscle allows quantification of fiber type specific gene expression.
abstract: Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature, and neurons, thus making it difficult to perform genetic or proteomic expression analysis with sufficient cellular specificity to resolve differences at the individual cell or myofiber type level. Here, we describe the combination of a simple histochemical method capable of simultaneously identifying Type I, IIA, IIB, and IIC myofibers followed by laser capture micro-dissection (LCM) to compare the expression profiles of individual fiber types, myonuclear domains, and satellite cells in frozen muscle sections of control and atrophied muscle. Quantitative RT-PCR (qPCR) was used to verify the integrity of the cell-specific RNAs harvested after histologic staining, while qPCR for specific genes of interest was used to quantify atrophy-associated changes in mRNA. Our data demonstrate that the differential myofiber atrophy previously described by histologic means is related to differential expression of atrophy-related genes, such as MuRF1 and MAFbx (a.k.a. Atrogin-1), within different myofiber type populations. This spatially resolved molecular pathology (SRMP) technique allowed quantitation of atrophy-related gene products within individual fiber types that could not be resolved by expression analysis of the whole muscle. The present study demonstrates the importance of fiber type specific expression profiling in understanding skeletal muscle biology especially during muscle atrophy and provides a practical method of performing such research.
authors.affiliation: Harvard NeuroDiscovery Center, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Vanderburg
authors.forename: Mark S F
authors.initials: MS
authors.lastname: Clarke
mesh_terms: D000818:Animals; D004396:Coloring Agents / Q000737:chemistry; D015870:Gene Expression; D006614:Hindlimb / Q000473:pathology; D019417:Hindlimb Suspension; D061346:Laser Capture Microdissection; D008297:Male; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism / Q000473:pathology*; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism / Q000473:pathology*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009133:Muscular Atrophy / Q000378:metabolism / Q000473:pathology; D009928:Organ Specificity; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods; D014048:Tolonium Chloride / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D009124:Muscle Proteins; D014048:Tolonium Chloride; D012313:RNA