Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA.
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.
Sun
Hai-Yuan
HY
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
McNally
Mark T
MT
Jackson
Vaughn E
VE
Grossberg
Sidney E
SE
D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D012313:RNA / Q000378:metabolism; D012335:RNA, Ribosomal / Q000032:analysis; D012367:RNA, Viral / Q000302:isolation & purification*; D012190:Retroviridae / Q000235:genetics*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014508:Urea / Q000378:metabolism*; D014771:Virion / Q000737:chemistry / Q000235:genetics*; D014773:Virology / Q000379:methods*
D012335:RNA, Ribosomal; D012367:RNA, Viral; D012313:RNA; D014508:Urea; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003851:Deoxyribonucleases; D012259:Ribonuclease, Pancreatic