RNA isolation of murine pancreatic islets - poor ratios - (Nov/23/2012 )

hello everybody,
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,
ceridwan

Hi Ceridwan,

I have ever worked with pancreas tissue. In my experience, I got good ratio of A260/A280 and A260/A230 and got high concentration of RNA , Its about 1400-2000 ng/µl. I used RNA later from ambion to save RNA in the tissue. For RNA extraction, I also have bad experience with trizol method. I got high concentration but poor ratio, then I tried to combine trizol with RNeasy method. And interestingly, although the RNA concentration slightly less than trizol method, the ratio seems almost perfect; I would recommend you try trizol combine with RNeasy. Just treat your cells with trizol until get aqueous phase, then transfer the aquoeus phase to a new tube, wash with 1x volume of 70%ethanol, and transfer it to the RNeasy column afterwards. Then just follow RNeasy protocol until finish. But keep in mind that in this method, just skip homogenization with RLT buffer because we will already done by Trizol. Beside that, before the last step with RNAse free water, just transfer column into new tube and sentrifuge with maximum speed for 1 minute to make sure there is no contamination by ethanol during the extraction process. This contamination will make the bad ratio.
Let me know if you get the better result

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.
cheers,
ceridwan