Hi All,
I was wondering if anyone had recommendations , insights, comments, etc. on what they think is the best RNA extraction protocol for small tissues. By small, I mean the brain of a tadpole that is ~3 mm cubed (the brain itself). We have a protocol that can yield ~8-10 ng/uL. However, I was wondering if others have had experience with similar sized tissues and if they have any recommendations. Any insights would be greatly appreciated!
Thanks,
Nick
Hi, do you know exact weight of your sample? (3 mm cube). Using standard column-based approach you should work with 20-40 mg of fresh tissue. It could give you good yield and quite good purity for subsequent reverse trancription. For me, the best choise is PureLink RNA Mini Kit.
I do not know about your goals, but in some cases it is necessary to receive more. In this case I always prefer phenol/chlorophorm based approach. You can try RiboPure™ Kit Family.
Thanks for your suggestion, Andrei S. Babenka ! We currently use Trizol + PureLink RNA Mini Kit because of the relative simplicity for training undergrads on the protocol. Our downstream application is for 3' RNAseq. I will look into the RiboPure Kits and see if that would be useful for us.
We work with tiny amounts of human tissue (leftover from diagnostic tests). For improving RNA quality, the best thing we have found is storing the tissue in an RNA preservative like RNAlater Stabilizing Solution. We get RIN scores above 8 this way (good for RNA-sequencing), whereas other attempts yielded poor results. See publication: Article Optimization of techniques for multiple platform testing in ...
For RNA extraction specifically, we homogenize the tissue with an extraction buffer containing 1% beta-mercaptoethanol, using RNase-free needles attached to a syringe. We go from 21G (largest diameter) to 25G (medium) to 27G (smallest), then put the homogenate through a QIAGEN kit like RNeasy Mini Kit or AllPrep DNA/RNA/(miRNA) Universal Kit. The former kit will be better for undergraduates (fast and easy), whereas the latter kit takes longer and isn't as undergrad-friendly (two columns, more buffer steps). If you just want total RNA >200 nt, I recommend the RNeasy kit. If you want matched DNA, then one of the AllPrep kits will work. We elute in 30-50 ul depending on the initial tissue size and get >50 ng/ul RNA elutions.
Good luck!
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