The samples were preserved at -80C, minced with sterile surgical blades in trizol (on ice) and proceeded with usual steps of Trizol extraction. Initially the pellet obtained was clear, easily dissolved in RNAse free water with A260/280 ratio in the range of 1.8-1.9 but when checked on gel, only one band was visible . To rule out degradation during electrophoresis run, another RNA sample extracted from blood was used as control which showed all the three bands.
After sometime, the RNA extraction was repeated, but now pellet obtained is light brown in color, not easily dissolved in water and A260/280 ratio in range of 1-1.3.
I've taken all the possible precautions, read many trouble shooting guides but still not able to figure out where the problem lies. Hoping for valuable comments over here
RNA extraction from skin tissue is challenging, firstly, the hyaluronic acid-collagen matrix is extremely difficult to homogenize. Second, skin tissues are almost always contaminated with nucleases. So, the success of RNA extraction from skin tissues lies primarily in the sample preparation, the skin sample must be frozen in liquid nitrogen or dry ice, and as such, Trizol as a lysis reagent is the right option. You can also try Betamercaptoethanol + Trizol. The second important thing is in the phase separation, where you don't need to be over ambitious for aq. phase recovery, which can definitely yield RNA with low quality (low A260/280 ratio), but if the phases separation is correct, your results may improve marginally. Good luck!
Have you ever tried Tri-zol based Direct-zol RNA Kits?
This kit isolate total RNA, including small RNAs from a variety of sample sources (cells, tissues, serum, plasma, blood, biological liquids, etc.).
Here is the link;
https://www.zymoresearch.com/collections/direct-zol-rna-kits/products/direct-zol-rna-miniprep-plus-kits
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