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Cite this page: Shackelford R. RNA purification. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurrnapurification.html. Accessed May 23rd, 2019.
Definition / general
- RNA is less stable than DNA, making its purification more difficult
- 4M guanidinium thiocynate or phenol with SDS are often used to denature RNases naturally present in blood, plasma, body fluids, bacteria, fungi, plants
- Must avoid sample contamination with RNases, which are extremely ubiquitous in environment
- Must use extremely clean reagents, such as diethylpyrocarbonate treated water
- Must keep samples cold to lower RNase activity; isolate pure RNA quickly and efficiently to minimize RNase exposure and avoid a basic pH in all extraction and storage solutions, as RNA rapidly degrades under basic conditions
Poly (A)+ RNA purification
- mRNA represents only 1 - 2% of total cellular RNA but is often analyzed for research and molecular diagnostic purposes
- Most eukaryotic mRNAs carry poly (A)+ 3' terminal tails, which can be used to isolate mRNA over an oligo (poly dT) attached to a solid matrix
- Under the right buffer conditions, cellular mRNA poly (A)+ tails will form RNA-DNA hybrids; the solid phase can then be washed to remove unwanted cellular continents and a later denaturing wash can be employed to remove pure mRNA
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