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Journal of Histochemistry and Cytochemistry, Vol. 47, 995-1004, August 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

RNA Content Differs in Slow and Fast Muscle Fibers: Implications for Interpretation of Changes in Muscle Gene Expression

Petra E.M.H. Habetsa,b, Diego Francoa, Jan M. Ruijtera, Anthony J. Sargeantb,c, José A.A. Sant'Ana Pereiraa,d, and Antoon F.M. Moormana
a Department of Anatomy and Embryology, University of Amsterdam, Amsterdam, The Netherlands
b Neuromuscular Biology Group, Manchester Metropolitan University, Alsager, United Kingdom
c Institute for Fundamental and Clinical Human Movement Sciences, Vrije Universiteit, Amsterdam, The Netherlands
d Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin

Correspondence to: Antoon F.M. Moorman, Dept. of Anatomy and Embryology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression. However, total RNA content per gram of a particular fiber type may differ as well. We have tested this possibility in the distinct fiber types of adult rat skeletal muscle. Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content. Quantification of the hybridization showed that the 28S rRNA content decreases in the order I>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type IIB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per µg of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis. Consequently, Type I fibers contain five- to sixfold more MHC mRNA per µg of tissue than IIB fibers. These differences are not reflected in the total fiber protein content. This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition. (J Histochem Cytochem 47:995–1004, 1999)

Key Words: skeletal muscle, single fibers, RNA content, in situ hybridization, densitometry


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