I am trying to isolate RNA from human adipose tissue but I am not able to get it. I have already tried from PBMCs and other cell type, from which I am able to isolate.
Please check the Tri reagent protocol on sigma website and pay attention to the notes, even you are not using their product, it should also work. Good luck.
http://www.sigmaaldrich.com/technical-documents/protocols/biology/tri-reagent.html
In my opinion the most crucial step for RNA isolation in general is the cleaning with phenol-chloroform an alcohol if it is done correctly you should get lots of RNA and with high purity.
Hi,
I use Trizol Reagent for both white and brown adipose tissues and primary cultures.
The most crucial step for RNA purity is when you collect the water phase without touching the white interphase. Also, in the last step, removing as much etanol as possible before dissolving the pellet in DEPC water is crucial for RNA purity.
Another detail that is not included in the Trizol is in the step when you add iso-propanol to precipitate the RNA. Most oftenly, two phases appear after iso-propanol addition to the collected water phase. I noticed that I didn´t get good amounts of RNA if I didn´t mix these phases. Therefore, I always briefly vortex the sample after isopropanol addition and have since then always had good amounts of RNA.
Hope this helped.
Cheers!
@Oscar Gabriel Sevillano Quispe: I am not getting RNA bands at all. While I am getting bands when I do RNA isolation from peripheral blood mononuclear cells.
Michał J Kowalczyk & Tomas B Walden: Thanks for suggestions and protocol. I will conducted by protocol given by you both.
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