This important research was part of the PhD work carried out by Petra Habets in the research group headed by Professor Anthony J Sargeant. It was a collaboration and jointly supervised by Anton Moorman of the Academic Medical Centre, of the University of Amsterdam. Sadly one of the inspirations for this research and close friend Jose Sant’Ana Pereira died, much too young, a few years after this work was published while working in the University of Madison, Wisconsin.
RNA content differs in slow and fast muscle fibers: implications for interpretation of changes in muscle gene expression
Petra E.M.H. Habets, Diego Franco, Jan M. Ruijter, Anthony J. Sargeant, José A.A. Sant’Ana Pereira, Anton F.M. Moorman.
Journal of Histochemistry and Cytochemistry
J Histochem Cytochem. 1999 Aug;47(8):995-100
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 microgram 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 microgram 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