Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR ( cetylcholine receptor and slow MyHC are...)

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

cetylcholine receptor and slow MyHC are amplified. The housekeeping gene GAPDH was amplified as a reference for the amount loaded and the quality of the cDNA. All primers (Table II) are intron spanning to produce a cDNA specific PCR product. To avoid false positive results by PCR product carry-over, PCRs are setup in a flow hood.

17. 15 l of each PCR product were loaded on a 2% agarose gel and separated by 120 V for 25 minutes. Gels are stained with ethidium bromide and visualized using the Gel Doc 2000^TM Gel Documentation System.

Results
The data presented in this application note demonstrates that it is possible to isolate a single muscle fiber type or a distinct muscle fiber population and analyze from it the expression of muscle specific genes by RTPCR. Figure 1 demonstrates the isolation of an individual fiber type by LCM. The Zenon^TMtechnology based immunohistochemical staining of slow MyHC positive fibers gives a strong, unambiguous signal in less than 35 minutes. The acetylcholine esterase stain of Karnowsky and Roots allows us to assess rapidly the innervation pattern of muscle fibers. When used alone, this esterase stain can provide results within 5-10 minutes. Hence, rapid identification of distinct fibers based on their immunohistochemistry and innervation pattern allows us time to process tissue via LCM and isolate mRNA without significant time for degradation.

Using this type of analysis on limb muscle confirms data previously obtained by other means⁴. Hence, we can apply the technique to another muscle allotype, the EOM, and be sure that this protocol and the Arcturus PixCell II Laser Capture Microdissection System is sufficient to distinguish muscle fiber types and give new insight into the differences among muscle allotypes.

References
1. Fischer MD, Gorospe
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( cetylcholine receptor and slow MyHC are...)Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR