Dear All,
I got 2 weeks old paraffin blocks of tumor masses. I want to extract total RNA from them. The available reagents in my lab are:
1) Cell dissociation buffer (GIBCO#13151-014)
2) TRI reagent ( Sigma#T9424)
Would these two reagents be effective to isolate RNA? Please need your help and suggestions. TIA!
Hello Nasir, how are you?
Well, it is not typically extract RNA from paraffin embedded tissues, but I think is possible. However, the quality and quantity of RNA will not be so good to analyze if you want RNA for qPCR or sequencing. If you trying to perform some of these techniques, I strongly suggest you get fresh samples to extract RNA with TRI reagent.
Nevertheless, for your current issue, you need to deparaffined your samples, remove the residual paraffin with xylene washes, remove the xylene with ethanol washes, and remove ethanol with distillated water washes. I am afraid that will degrade your RNA sample.
After these steps, you can try to use your Cell dissociation kit to divide your sample and use the TRI reagent for isolate RNA. If you will dissociate your sample, try to get high quantity of cells per tube. Alternatively, you can fragment your sample with a scalpel and try to extract RNA without dissociation cell kit. Good luck.
Túlio Teruo Yoshinaga I am good. Thanks! Hopefully you are also doing good.
I will follow your suggested procedure. It's really helpful.
Is it possible that some of the cells will be lost during washing steps? In this case, do I need to centrifuge sample after each wash to stop loses?
I'm doing well... Thank you. If your embedded sample is not dissociated in cells, in case, your tumor samples still in a whole piece, you do not need to centrifuge. Centrifuge will help eliminate paraffin, xylene, and residual ethanol in case you sample is already dissociated, but I think it's not your case.
Thank you. After xylene and ethanol washes, I could still see the paraffin chunks so I didn't perform cell dissociation but added 1 ml TRI reagent to each tube and incubated at 60C O/N. Today I will perform next steps of RNA isolation.
I think I screwed it up. Did I? :(
Hum, if there is some paraffin left, this could affect RNA isolation. Probably you should washed your samples more time. Maybe you won't get a good RNA or high quantity. In situ hybridization techniques is for visualize RNA in parraffin sections. So, there is some RNA fixed by formalin. But again, try to get some fresh samples... If you don't have RNA later fixative solution, you can store in Tri Reagent at -80°C until processing.
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