Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA. J Virol Methods 2006 Nov;137(2):304-8
Date
08/22/2006Pubmed ID
16920200Pubmed Central ID
PMC2575689DOI
10.1016/j.jviromet.2006.07.003Scopus ID
2-s2.0-33748714112 (requires institutional sign-in at Scopus site) 1 CitationAbstract
Cellular nucleic acids can interfere with the molecular cloning of retroviruses, a problem that is particularly serious with viruses propagated in lymphoblastoid cells that release large amounts of microvesicles and other cellular components. The approach taken to circumvent such problems involved first suspending viral pellets in water to allow any residual microvesicles to swell and perhaps lyse during overnight or longer incubation periods. Urea was then added to a concentration of 1.5-2.0 M to uncoil proteins that may protect nucleic acids from hydrolysis on the further addition of Micrococcal nuclease and ribonuclease A, both of which remain enzymatically active in molar urea solutions. The viral RNA was extracted and residual DNA removed by deoxyribonuclease I treatments. The utility of the method was demonstrated with two different retroviruses, a Moloney murine leukemia virus variant and Rous sarcoma virus, such that viral RNA thus purified was shown to be free of contamination by PCR-amplifiable cellular GAPDH mRNA and ribosomal RNA. This general approach should be applicable to viruses of any type in circumstances where contamination by cellular RNA and DNA poses a problem.
Author List
Sun HY, McNally MT, Jackson VE, Grossberg SEAuthor
Mark T. McNally PhD Associate Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
DNADeoxyribonucleases
Glyceraldehyde-3-Phosphate Dehydrogenases
RNA
RNA, Ribosomal
RNA, Viral
Retroviridae
Ribonuclease, Pancreatic
Urea
Virion
Virology
