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Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR

Sven Fraterman and Neal Rubinstein, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia


Abstract
To locate gene products in distinct muscle fiber populations of different muscle allotypes, a novel protocol was established. A rapid method to distinguish muscle fiber types using histochemistry and immunohistochemistry was used to provide criteria for their selective isolation by laser capture microdissection while preserving messenger RNA (mRNA). Polymerase chain reaction showed a differential expression pattern of muscle specific genes in different muscle fibers in laser captured material.


Introduction
While vertebrate limb muscles have traditionally been the paradigm for studies of skeletal muscle myogenesis, a few atypical muscle groups such as extraocular muscles (EOMs) have distinct functional specializations and patterns of gene expression1. Differences between muscle groups (called allotypes) have been the subject of research for the past decades. The limb and EOM allotypes differ in their pattern of innervation. All limb fibers and 80% of EOM fibers are singly innervated fibers (SIFs) which have only one neuromuscular junction. About 20% of EOM fibers are multiply-innervated fibers (MIFs): they have multiple neuromuscular junctions2. In the past, immunohistochemistry and in situ hybridization were mainly used to study differences between muscle fiber populations3,4, While useful, these techniques are limited in their capability to assess multiple gene products from a mixed muscle fiber population. Moreover, the mixed fiber populations of these muscles make it impossible to study the composition of individual fiber types. To overcome these problems, laser capture microdissection was used to isolate different muscle fiber populations and analyze multiple gene products by polymerase chain reaction
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