Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR ( acetylcholine esterase stain and their ...)

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

acetylcholine esterase stain and their lack of slow MyHC staining (Figure 1C). MIFs were dissected based on their smaller neuromuscular junction and a positive signal for slow MyHC (Figure 1A). Additionally, muscle fibers were dissected which were positive or negative for slow MyHC, but didnt show a site of innervation based on Karnowsky and Rootss acetylcholine esterase stain. To dissect single fibers, it can be advantageous to search for fibers on the edge of a fiber bundle as they prove to be easier to dissect without picking up undesired fiber populations.

13. To avoid cross-contamination by other cell types, every cap is scanned visually after the dissection by placing the cap on a fresh slide and viewing it at lowest power. This is followed by a scan at the highest power to make sure that only the desired tissue was captured based on the fluorescence signal (Figures 1B and 1D). The cap is discarded, if unwanted tissue was present. Monochrome pictures of samples were taken using the QImaing^TM Retiga 1300 cooled mono 12-bit digital camera.

14. Each cap is then placed in an ExtracSure^TM device. The RNA from approximately 25 fibers of each population is pooled and RNA extraction is performed with the PicoPure^TM RNA Isolation Kit as described in its protocol within 2 hours of the dehydration with xylenes.

15. After the RNA is extracted, the RiboAmp^TM RNA Amplification Kit is used according to the manufacturers instructions to linearly amplify the sample RNA and transcribe the aRNA back to single stranded cDNA. One round of linear amplification was performed for all samples, typically yielding 300 - 600 ng of aRNA.

16. To study expression of muscle specific genes, 50 ng cDNA are amplified with FastStart Taq DNA Polymerase for 40 cycles with conditions and reagents as presented in Table I. The epsilon- and gamma- subunit of the a
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( acetylcholine esterase stain and their ...)Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR