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Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein

d or fluorescence) during LCM allows accurate capture of EGFP-expressing cells

7. After completing LCM, perform extraction, purification and amplification of RNA using the Arcturus PicoPure and RiboAmp kits according to the manufacturers protocols.

8. Synthesize cDNA from amplified RNA with random hexamer primers and Superscript II reverse transcriptase to confirm expression of cell type-specific gene targets by quantitative real-time PCR (Bhattacherjee et al., 2004).


Results
During dissection, embryos in a litter that express EGFP in a pattern specific to neural crest cells can be identified by fluorescent stereo microscopy prior to fixation and embedding in OCT (Figure 1). EGFP-labeled neural crest cells can be identified and captured by LCM from partially fixed cryosections of embryonic tissue (Figure 2). Quantitative Real-Time PCR of the RNA extracted from these captured cells resulted in amplification of neural crest cell gene markers (Crabp1, Dlx5, Msx1, Slug), while expression of Engrailed-1 (En1), a gene that is not expressed in the neural crest cells was not detected (Table 1). These results demonstrate the utility of LCM to isolate specific EGFP-expressing cell populations from tissue sections of transgenic mouse embryos that can be used for subsequent molecular analyses.



1Expression of neural crest gene markers was determined using TaqMan quantitative realtime PCR.
2+ indicates detectable levels of gene expression; - indicates undetectable levels.
3Neural crest cDNA samples were prepared and subjected to QRT-PCR for each target gene in triplicate; mean Ct values are reported.
4Negative methodological control reactions, which lacked reverse transcriptase, did not amplify any detectable product.
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