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Total RNA extraction from your samples cells line by using the Qiagen RNeasy Mini Kit
To extract the RNA from a tissue or a cell suspension, it is imperative to inhibit traces of RNase.
Homogenization in the Potter tube is done in a Tris-EDTA buffer in the presence of 4M guanidinium thiocyanate and SDS. Cellular debris is pelleted by 10 min at 10 ° C.
The supernatant containing the nucleic acids is deposited on a density gradient tube comprising at the bottom of a dense CsCl solution topped 5.7M of an intermediate layer of 2.4 M. This CsCl gradient was ultracentrifuged for 24 hours at 30000 revolutions / min at room temperature.
We can then collect a high molecular weight RNA pellet will be subjected to extraction with phenol and chloroform.
The key in RNA isolation is to rapidly deactivate RNases, the enzymes that quickly degrade RNA. The problem with RNases is that they are found everywhere in living cells/tissues and in the environment. Some methods of RNases deactivation include quick addition of SDS and guanidine salts to the sample and/or freezing with liquid nitrogen.
As for separation, a non-polar organic phase (like phenol) is added, the mixture centrifuged, and aqueous phase (containing RNA) is recuperated and precipitated by ethanol.
Alternatively, if you have the resources, buy an RNA extraction skit from a scientific company, (such as ThermoFischer) and follow their instructions!
RNA extraction kit is the best and accurate way to obtain RNA with high purity as the kit is already tested for it..
By using the Northern blotting and RNAase enzyme we can extract the RNA from the sample. Because the role of RNAase enzyme is degrade the RNA.
Rapidly deactivate RNases which is responsible for degradation of RNA.
Make sure everything (Pipette, tips,glassware, gloves) is contamination free as RNA is highly unstable and gets degraded easily.
plasmid DNA purification kit ,extraction of DNA from cells , tissues and other
So that we can obtain ARN we do not have an ADN to be pure free from mutations
by excissing
elimnation of exons et collage of introns
The purest RNA is obtained by conventional (phenol / chloroform) methods based on the procedure described in: Anal Biochem. Apr; (1):-9. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Chomczynski P, Sacchi N.
or with more recent reagents such as Trizol (based on a similar principle). After isolation, it is necessary to treat dissolved RNA with the DNAase enzyme, and check the integrity on AGILENT Bioanalyser. Take care about RNase-free environment. If you want to archive a portion of the isolated samples, you can store aliquots of the isolates precipitated under ethanol for years
