question about LCM

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      johnmay
      Participant

      Hello all,
      Recently I am set to perform LCM on PFA-fixed midbrain sections of TH-GPF transgenic mice and encountered some problems. The quantity and quality of RNA isolated from LCM cells were too poor to do the downstream analysis like real-time PCR. Because the GFP expression in the cells of interest can help visualize the cells, I don’t have to perform a staining step. Also, I didn’t do the dehydration step since the alcohol could quench the GFP signal remarkably. Then it was difficult for us to capture a single cell using IR laser on LCM machine, what we can do was just to use UV laser to cut the cell patches, which was not we want to do.
      (1) Is the dehydration step very important for cell capture? If yes, how can I perform this step? (the literatures refers the graded ethanols and xylene, but ethanol could quench my GPF signal)
      (2) What kind of slide is better for single cell capture by IR laser? Glass or membrane? And the thickness of the section? I use Arcturus PicoPure RNA Isolation Kit (#KIT0204) to isolate RNA from LCM cells, is it better to add proteinase K to the Extraction Buffer to help extraction since the proteinase K could help extract RNA by reversing the extensive crosslinking network of aldehyde?

      Any reply will be highly appreciated,
      Zhong

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