We are patching neurons followed with extracting their cytoplasm to do single cell rt-pcr analysis.
The controls we do are to stick the patch pipette into the tissue, to mimic approaching a cell, and to suck extracellular fluid around the cells.
All controls are coming back positive with RNA.
How do we avoid this? Does anyone have any experience with this?
Dear Elizabeth,
I suggest you also look at the controls in the publications from James Eberwine's lab at U Penn, or Diane O'Dowd at UC Irvine. I do not have access to PubMed at the moment, so I can't get you the PDFs.
Unfortunately, I have only tried this in vitro, so it is much cleaner and easier to patch the neurons.
Good Luck!
Jill
Dear Elizabeth,
It may be that it is difficult have negative controls with sucking the extracellular fluid that is probably full of various RNA molecules due to tissue damage and leakage in to the extracellular fluid/matrix during slicing and electrode penetration. Given the sensitivity of amplification process, even a few copies will give you positive results. Therefore it may be better to suck into your electrode just the bath solution, without going into the tissue. Another option will be to do PCR of dissociated cells. We have in the past succesfully performed single cell RT-PCR for TRPC channels in isolated/dissociated hair cells from the cochlea. For details please see the following publication.
Best wishes, Refik
Article TRPC-like conductance mediates restoration of intracellular ...
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue