Internally controlled quantitative assays for PKR mRNA and protein from liver biopsies and other finite clinical samples.
A method to quantify double-stranded RNA-dependent protein kinase (PKR) mRNA and protein from human cells is described. A competitive RT-PCR assay has been developed by synthesis of an internal standard control (ISC) species of RNA. A competitive immunoblot assay was used to quantify full-length PKR (FL-PKR) protein in a sample of total cellular proteins, using truncated PKR protein as an internal standard against which FL-PKR protein could be quantified. The method can be used for simultaneous analysis of transcriptional and postranscriptional regulation of PKR gene expression from very small clinical specimens such as liver biopsies, e.g., 2-3 mg (wet weight) and containing only 2x10(5) cells. To the best of our knowledge, this is the first report of a sensitive simultaneous assay system for this important immunoeffector molecule.
Farrugia
J
J
Department of Microbiology, University of Leicester, Medical Sciences Building, University Road, Leicester, UK.
Cann
A J
AJ
D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015151:Immunoblotting / Q000379:methods / Q000592:standards; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D019892:eIF-2 Kinase / Q000032:analysis* / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D019892:eIF-2 Kinase