Translational extracts active biologically in vitro obtained from eukaryotic monolayer cells: a versatile method for viral RNA studies.
Preservation of enzymatic activities in biological samples, especially after freeze/thawing, is a crucial requirement in virological research. Theoretically, this preservation can be achieved with the presence of cryopreservative agents. In contrast to tedious methods, it was found that this might be readily achieved by using well-defined conditions, including sucrose in the samples. Hence, the generation of a translational extract obtained from eukaryotic cells that have grown as monolayers is described below. This versatile method could be used advantageously for the in vitro translation of messenger RNAs, added exogenously, including viral mRNAs. The translational extract can be prepared freshly on a daily basis, or more conveniently it can be frozen and thawed subsequently for further use, without loss of activity. It can replace the Krebs ascites fluid and the commercial rabbit reticulocyte lysate. The procedure employed for the preservation of the biological activity of the translational extract can be extended to various other biological samples.
Favre
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Unité de Recherche sur les Virus des Hépatites, Institut National de la Santé et de la Recherche Médicale, Unité 271, 151 Cours Albert-Thomas, 69003, Lyon, France. favre@lyon151.inserm.fr
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D000818:Animals; D002457:Cell Extracts; D002478:Cells, Cultured; D006224:Cricetinae; D015925:Cryopreservation; D003593:Cytoplasm / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D002457:Cell Extracts; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D013395:Sucrose