I am Trying to extract high-quality RNA from acinar pancreatic cells after collagenase P digestion and FACS sorting.
Can you give me some advises to obtain no-degraded RNA suitable for RNA seq?
Thank you
It is exceptionally difficult to get good quality RNA from pancreatic acinar cells even under optimal conditions, but it can be done. The issue here is that acinar cells, themselves, produce a huge amount of RNAse.
The only way I could imagine this working is for you to submerge the tissue in RNAlater thoroughly prior to collagenase treatment. Maybe even see if you can do the collagenase in RNAlater?
But, the larger question is: why do you need to do FACS sorting? Can you just isolate a piece of pancreas and submerge it in RNAlater? If your concern is contamination by islet cells I wouldn't worry much about it; there is FAR more acinar content than islet content in the pancreas.
Remember, for stabilizing (RNAlater, TRIzol), if a protocol says to use 10 volumes for a normal tissue, use more like 100 volumes for pancreas. The RNAse is pervasive.
Yes I thought that incubating the tissue with RNAlater before starting digestion could be feasible to improve the quality of RNA, I will try it soon !!
I need FACS sorting because I am working on transgenic mice and I need to select YFP+ acinar cells bearing that mutation. I know, the idea is very ambitious, especially in the pancreas but based on our preliminary results, I think it could be achieved...
Would it be an option for you to have pancreatic tissue and to isolate fluorescent cells selectively by laser microdissection instead of preparing a solution of cells?
I need a maximum of cells with good RNA quality. For micro-dissection the tissue needs to be fixed and paraffin-embedded and this will give a limited quality of RNA; it is better to use fresh tissue. Indeed, another colleague is working with micro-dissection on liver to isolate a small population of cells and he faced much problems.
That's true for most laser microdissection systems. To my knowledge, MMI is the only provider of LMD systems, who also offers Live Cell Chambers to cut living cells. Please find the corresponding protocol attached.
Would this procedure be an option for you?
http://www.molecular-machines.com/libraries.files/Live_Cell_Microdissection321.pdf
I am coming back few months later with the answer.... For all those interested with this kind of experiments herein the link to our protocol paper in which we describe the method to isolate acinar cells and extract high quality RNA. Don't hesitate to read the paper and post ur feedback:
Article DIE-RNA: A Reproducible Strategy for the Digestion of Normal...
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