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) mRN A 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 P KR protein as an internal standard against which FL-PKR protein could be qu antified. The method can be used for simultaneous analysis of transcription al and postranscriptional regulation of PKR gene expression from very small clinical specimens such as liver biopsies, e.g., 2-3 mg (wet weight) and c ontaining only 2 X 10(5) cells. To the best of our knowledge, this is the f irst report of a sensitive simultaneous assay system for this important imm unoeffector molecule. (C) 1999 Elsevier Science B.V. All rights reserved.