In RNA extraction we are not expecting to break the nuclear envelope.So, there is no need to use a strong lysis solution.
What is the main difference between DNA or RNA extraction?
What should the pH levels be?
RNA is very sensitive to RNAses. Guanidinium is a very strong denaturing agent so that it both dissolve cells and denaturates endogenous RNAses.
Differences would be different, depending on what type of RNA you want to extract.
Remember RNAases are Proteins which are the most stable proteins. You need strong denaturants such as Guanidium. This will destroy the RNA degrading enzyme so that you could get good quality RNA. Whereas when you are isolating DNA you need to worry about DNA degrading enzyme (DNAase) which are relatively easy to degrade with reagent such as phenol. Best of wishes.
in my opinion,,. rna extraction is much more delicate than dna extraction because of the edlicate nature of rna. so prime difference between the procedures are that rna extraction is sensitive to any impurity or temperature much more than the dna. of all the impurities, rnases being the most hazardous ones. rnases are all around us and we consistently secrete them through our skin as well. this high abundance of rnases in the enivironment demands a high efective rnase destroyer like guanidium chloride (or guanidium thiocyanate) which destroys not just rnases but most protein by inverting their structures and adduct formations. the proteins which were folded in a aqueous environment in cell get reversed when in presence of guanidium thiocyanate. that is all i know.. hope it helps. and also you may want to break the nuclear envelope based in the rna you are looking for. extraction process changes based on the different classes of rna you are planning to extract.
Hi, I have an identical issue. while trying to isolate RNA from orchid roots I am referring a protocol where guanidine sulphate is used. But in my lab I donot have at present. Instead I have Guanidine isothiocyanate and guanidine hydrochloride. Can i supplement it with any one of them?
RNA lysis buffers that contain guanidinium thiocyanate or guanidinium–HCl reproducibly yield very high-quality RNA samples. This is true because of the extremely chaotropic nature that these chemicals exhibit; they are among the most effective protein denaturants (Cox, 1968; Nozaki and Tanford, 1970; Gordon, 1972)
https://www.sciencedirect.com/science/article/pii/B9780123747273000024
RNA Isolation Strategies
Robert E. Farrell Ph.D, in RNA Methodologies (Fourth Edition), 2010
I need to do whole genome DNA extraction from pellets of deer family. The tissues are quite hard. I tried various conventional methods for DNA extraction, but the results were not good. However, I found one method in which lysis buffer Gu-thiocyanate is used, but I do not have it my lab. Can I use GuHCL instead for lysis buffer.
Guanidium is a strong reagent. Are there any possibilities of RNA denaturing by guanidium?
In fact you can you either for RNA or DNA extraction. The thiocyanate salt is a stronger chaotropic an is preferred for RNA, to degrade the RNAses. For RNA you use the HCl salt because it is cheaper. The chaotropic makes the nucleic acid bind to the silica in a column, or if you do buffer-based extraction without column it separates with the nucleic acid in the inorganic phase, which you then wash out with diluted ethanol. The strength the lysis solution is not about the nuclear envelope, but about the RNAses that float around.
Main difference between RNA and DNA extraction is their stability. RNA is more labile and gets degraded by RNAses very quickly and efficiently. To avoid degradation you always work on cold samples: liquid nitrogen or dry ice to grind, ice to extract. You do the extraction with guanidine buffer, or phenol buffer, or phenol-guanidine buffer, or CTAB, or CTAB-chloroform. In the end, the goal is to destroy the RNAses before they degrade your RNA, and then separate proteins, lipids and carbohydrates from RNA to get your RNA clean. You use pH differences to separate RNA from DNA in the different organic phases in your extraction (eg with you use Trizol, which is phenol_guanidine), or after nucleic acid purification you get rid of DNA with DNAses and follow up with column purification or precipitation. Then RNA you can precipitate differentially from DNA using a lithium salt.
DNA you can extract with a Tris-based buffer, and you only get concerned a bit by DNAses. These you inactivate with heat and/or EDTA, because they are quite labile. The rest is about again separating your nucleid acids from the rest, with the buffers above. Usually you get rid of RNA by RNAse treatment.
Get a look at the Sambrook-Maniatis book, or at the Current Protocols series, they are fantastic books to understand these things. You are asking a super-broad question, so those books will really help!
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