We are planning to extract RNA from mice tumors. Our downstream application will involve doing quantitative PCR (from cDNA). Does anyone have experience what is the best protocol of RNA isolation for this purpose? I have used Qiagen RNeasy kit for RNA isolation from cells (with on column DNAse digestion), and it worked well for me. For large chunk of tissue (in this case, tumor), what is the ideal procedure for collecting the tissue, and processing it (homegenization)? Is fresh tissue better or could it be flash frozen and saved in -80C and then thawed on the day you are performing the RNA isolation procedure?
If someone has a protocol and you would like to share, that'll be great. Thank you.
- The same kit i.e. RNeasy kit (Qiagen) will work for isolating RNA from tissues.
- Tissues should be collected on ice, washed in Phosphate buffer saline pH 7.4 until free from blood or debris.
- For storage, tissues should be dipped in RNAlater RNA Stabilization Reagent (e.g. Qiagen Cat No.: 76104 ) and kept at -80°C until analyzed. For isolating RNA, use liquid nitrogen, and use mortar-pestle for crushing the tissue. Make sure your tissue is crushed properly (in liquid nitrogen when it looks like powder form, transfer quickly in eppendorf without getting it thawed). Then proceed for RNA extraction steps.
Thank you for your answer, Bhairavi. Just one question - if I am able to flash freeze the tissue piece(s) in liquid Nitrogen right away, do I still need to use RNA later for saving purpose?
Quick freezing of tissues preserves RNA. There is no need to add RNAlater if you are immediately freezing in liquid nitrogen. Just try to avoid freeze-thaw cycles.
Keep tissue frozen. This prevents breakdown of RNA by endogenous RNases and preserves the expression pattern of RNA within the sample.
There is RNAlater recipe if you want to try,
https://www.protocols.io/view/RNAlater-Recipe-c56y9d
you can do a test-run with similar tissue treated by both ways to see if you can get comparable RNA in both tissues (Freeze-thawed / RNAlater).
However i have no experience with that, I used commercially available RNAlater.
Better trying RNAzol from sigma. Find the attachment for further details.
Atreyi Ghatak
For the extraction and isolation, if you want a cheaper option than Sigma or Qiagen, you could try the Quick-RNA kit from Zymo.
https://www.zymoresearch.com/collections/quick-rna-kits
Or if you're using Trizol/TRIreagent there the Direct-zol RNA kits
https://www.zymoresearch.com/collections/direct-zol-rna-kits
For preserving/stabilizing RNA at ambient temperature for >30 days there's DNA/RNA Shield
https://www.zymoresearch.com/pages/dna-rna-shield
MagMax mirVana kit efficiently recovers total RNA (including small RNA fraction) from tissue and many other sample types- manually or automated on KingFisher
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