Molecular Pathology - RNA Purification

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Molecular Pathology

DNA Purification

RNA Purification

Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2012, PathologyOutlines.com, Inc.

General
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● 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
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● 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

End of Molecular Pathology > DNA Purification > RNA Purification

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