Direct analysis of RNA after transfection.
It is possible to transfect mammalian cells with a gene of interest and directly detect the RNA made from that gene 24 to 48 hr later. The ability to analyze the RNA directly allows mutation and functional analysis of an intact gene, as the promoter does not have to be subcloned. This technique is also essential when fusion genes are used. In order to be sure that the level of the reporter protein produced by a fusion gene provides an accurate measure of appropriately initiated RNA from the promoter under study, it is necessary to determine the amount and 5' end of the fusion message. An investigator who verifies this using direct RNA analysis can proceed with some confidence that the level of the reporter protein is a measure of promoter activity. The difficulty in directly analyzing RNA is that the sensitivity of detection is at the limits of present technology. Consequently, the transfection protocol, RNA preparation, and RNA analysis techniques all have to be working near optimum in order for an experiment to work. This overview discusses parameters that must be considered when directly analyzing RNA after a transfection: transfection efficiency, the method of preparation of the RNA, the method of analysis of RNA, and strength of the promoter.
Kingston
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Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
D000818:Animals; D005822:Genetic Vectors; D006801:Humans; D007202:Indicators and Reagents; D008322:Mammals; D009154:Mutation; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D012260:Ribonucleases; D014162:Transfection / Q000379:methods*
D007202:Indicators and Reagents; D012367:RNA, Viral; D012260:Ribonucleases