Methods
DNA / RNA purification
RNA purification
Copyright: 2008-2019, PathologyOutlines.com, Inc.
PubMed Search: RNA purification[TI]
DNA / RNA purification
RNA purification
Topic Completed: 1 April 2012
Minor changes: 26 December 2019
Copyright: 2008-2019, PathologyOutlines.com, Inc.
PubMed Search: RNA purification[TI]
Cite this page: Shackelford RE. RNA purification. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurrnapurification.html. Accessed May 30th, 2020.
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
