Hi all, I am having trouble extracting RNA from frozen breast cancer tissue.
I added Trizol I felt that the quantity of RNA will be abundant(100mg tissue/1mL Trizol and the solution felt sticky), however when I added isopropanol and precipitate at 4℃ (trying -70 ℃ overnight now but I don't know if it will work) then centrifuge, some of the tubes had no RNA pellet visible, others have only petite RNA pellets(in my experience with blood white cell samples of same amount there should be a large RNA pellet), the upper phase of chloroform was a bit yellow(which should be totally colorless), I am not sure if its the lipids in the breast tissue at work here.
Breast cancer tissue was resected then kept in liquid nitrogen until use, I am using the protocol to extract RNA from cultured cells and use a tissue homogenizer to crumble the tissues before adding chloroform.
I have googled this and someone suggested RNeasy lipid tissue kit(Qiagen), I want to check up with more people before I have to buy a new kit.
Thanks!
Using commercially available kits from a number of companies for RNA extraction all work well. You have to keep in mind the ratio of Trizol reagent used to tissue remains the same. Using insufficient reagent will result in less RNA extraction. I would suggest first pulverizing the tissue in liquid nitrogen before adding the Trizol reagent. This would help in solublization of the tissue and increase the RNA yield. Pelleting RNA at -70C overnight does help but remember to centrifuge at 4C for a little bit longer than usual. Hope it helps.
Few thing I have to add
1. crush your tissue with Trizol with a micro pestle until the red trizol color turned into pink and solution to sticky
2. wait for 2- 3 minute in RT condition
3. centifuge at max, and 4 degree condition take the sup in a new vial.
4.add chloroform and shake vigorously for 10-15 sec.
5.wait for 10-15 sec @ RT.
6.centrifuge @ 4 degree
7.separate the the sup in a new tube
8.add 500-750 uL Isoprop, volume of isoprop depends upon the whit ring formation at the junction of two solution.
9. wait to react for 15-20 sec
10. keep inside -80 for over night
rest are the same but after adding any solution during isolation of RNA wait for few seconds for every steps to react.Another thing do not over dry the RNA pellet after 70% ethanol wash and store in a RNA storage solution @ -80.
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