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Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions

min). The reaction products are separated on a 2% Nusieve 3:1 agarose gel (Fig. 3B) to verify the appropriate size of the amplicons (~63- 67 bp; see Special Considerations, Note 5).


Results
To aid in the identification of foam cells for laser capture, aortic sections were immunostained for the CD68/Macrosialin antigen (red color; Fig. 1). RNA extraction of the laser captured material yielded 3.520.18 ng of total RNA per animal (meanSD). The integrity of the RNA was assessed and was found to be of high quality (Fig. 2). Peaks corresponding to the 28S and 18S ribosomal RNAs were clearly visible on the BioAnalyzer profile (A) and pseudo-electropherogram (B). There was no detectable genomic DNA contamination, as evidenced by a lack of additional peaks to the right of the 28S peak. Typically, RNase degradation of total RNA samples produces a shift in the RNA size distribution toward smaller fragments and a decrease in fluorescence signal and the 18S and 28S peak can no longer be identified with certainty. Degraded total RNA will lack a smooth baseline and typically contains multiple peaks that are as large as or larger than the ribosomal peaks. For the different gene transcripts measured, the quantitative RT-PCR assay was highly reproducible (median intra-assay coefficients of variation, based on >20 samples run in duplicate, ranged between 2.4% and 7.7%) and highly sensitive (10 pg of starting total RNA resulted in detectable product formation). In Fig. 3A, representative standard curve plots for CD68 (top) and αactin (bottom) are shown. For all of the genes measured, a linear relationship between the log of the initial RNA concentration (pg) and the threshold cycle held true over a range of five orders of magnitude variation in the starting RNA concentration (for all experiments, the correlation coefficients were between 0.980-0.999). RT-PCR products from the
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