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RNA purification


Rodney E. Shackelford, D.O., Ph.D.

Topic Completed: 1 April 2012

Revised: 15 February 2019

Copyright: 2008-2018, PathologyOutlines.com, Inc.

PubMed Search: RNA purification[TI]


Rodney E. Shackelford, D.O., Ph.D.
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Table of Contents
Definition / general | Poly (A)+ RNA purification
Cite this page: Shackelford R. RNA purification. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurrnapurification.html. Accessed July 19th, 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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