RNA isolation of murine pancreatic islets - poor ratios - (Nov/23/2012 )
i have to isolate total rna of pancreatic islets for performing microarrays. my first trials with trizol (100 islets in 1ml) or nucleospin II RNA (100 islets) resulted in poor ratios (A260/A280 and A260/A230). for rna isolated with trizol i used the nucleospin II as clean up. thus i do not have a lot of experience with isolating rna i am asking myself, if one can use too much trizol reagent for sample homogenization, how i can improve my rna ratios and if there is something special to take in account isolating rna from murine pancreatic islets (isolated by common collagenase perifusion of the entire pancreas).
i would really appreciate your help,
have a nice evening,
I have ever worked with pancreas tissue. In my experience, I got good ratio of
Pancreas cells are full of RNases so any protein carry-over will be a problem for the extraction. You need to ensure that the top phase at the chloroform step doesn't contain any of the white layer protein layer, you may have to leave some of the phase behind to achieve this, but as you do this more and get better at it, you should be able to get most of the phase into the rest of the extraction.
If you use a lot of trizol relative to the amount of cells you will get a low level of RNA extraction. Check the trizol manual/product sheet for information on roughly how much tissue or cells to use per volume.
thanks a lot for your help. i'll try what you told me and see if i can get better results.