Isolation of the high quality RNA is the first step for numerous molecular biology experiments. These experiments can be RT-PCR, Real-Time PCR, cDNA library construction, Northern blotting, RNA mapping, in vitro translation, etc. Here, in this article, we will deal with detailed protocol of RNA isolation.
The biggest rival of RNA during the extraction procedure is RNases. In RNA, hydroxyl groups are present at 2' and 3' positions of ribose sugars. This makes RNA much more chemically reactive than DNA. Therefore, contaminating RNases easily cleaves the RNA.
2 comments:
I am doing a RNA isolation presently, I have 3 doubts 1)instead of Trizol I am using a denaturating buffer containing 0.4M Urea, 25mM Sodium acetate and 0.5% of lithium chloride do you think it will be strong enough. 2)As in DNA isolation should I use a cut tip in places. 3)What will be the effect on yield if I don't use a 4*C centrifuge?
Vino, we generally use extraction buffer having chaotropic agents. However, we haven't used the buffer quoted by you. Therefore, we can't comment on the productivity of the above mentioned buffer. Generally cut tips are recommended for DNA isolation because of the larger size of the DNA molecules. However, in RNA isolation there is no need to use cut tips. You should always maintain 4*C temperature as it prevents RNA degradation, resulting in good yield and quality of the isolated RNA. If you have further queries, please feel free to ask.
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