We are having trouble looking up at the cellular source of our interleukin by regular intracellular staining by flow cytometry.
We'd like to try to detect the mRNA in our cell of interest from mice.
We need to gate our cells by flow cytometry by using a transcription factor as a positive marker.
After cell sorting, will the fixation and permeabilization steps interfere with a RT-PCR protocol (RNA extraction/purification, cDNA synthesis,...).
Thanks in advance for your advices.
alexis
Hi Alexis
The whole FACS protocol can compromise your mRNA and the initial steps for qPCR. I recommend you use a RNA extraction kit suitable for FFPE. Some kits can be used for fixed cells eventhough they are not paraffin embedded. But again, this will only help if your FACS protocol didn't damage your mRNA. Take a look at the article bellow
http://www.ncbi.nlm.nih.gov/pubmed/23434645
Hi Alexis,
I am not sure, if you still need suggestion regarding your question, but, I am working on somehing similar and we have recently came acroos the attached article and we are following it for our project. If it suits your experimental design, then you can give it a try.
Hi Alexis,
you can try this protocol "Fixed single-cell transcriptomic characterization of human radial glial diversity"
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