A novel method for quantitation of human von Willebrand factor messenger RNA.
We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700 quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism BigDye terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r(2)=0.912, p<0.001).
Kerr
Ron
R
Department of Haematology, The Royal Infirmary of Edinburgh, 1 Lauriston Place, Edinburgh EH3 9YW, UK. Ron.Kerr@ed.ac.uk
Stirling
David
D
Rae
Marion
M
White
Anne R
AR
Ludlam
Christopher A
CA
D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D012016:Reference Values; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014841:von Willebrand Factor / Q000235:genetics*
D012333:RNA, Messenger; D014841:von Willebrand Factor