Analysis of glycoprotein Ia, Ib, IIb and IV RNA in platelets: quantitative determination using fluorescence-based...

The aim of this study was to analyze the RNA level of glycoprotein (GP) receptors in platelets. We have therefore established a quantitative fluorescence-based polymerase chain reaction (PCR) to analyze GP Ia, Ib, IIb, and IV RNA. Isolation of platelet RNA was performed by guanidium isothiocyanate/phenol chloroform extraction. An internal standard consisting of cRNA copies from plasmid pAW109 was included before reverse transcription in each RNA sample and PCR amplification was performed using fluorescence-labeled primers. Subsequently, PCR fragments were separated by gel electrophoresis and quantitation of the GP-specific fragments was done by measuring the fluorescence intensities in comparison to the internal standard. Relative amount of GP RNA/platelet were calculated taking into account the number of platelets used for isolation of platelet RNA and the platelet size as determined by flow-cytometric analysis. Using this method we analyzed the GPIa, Ib, IIb and IV RNA content of platelets in healthy blood donors. In parallel experiments the number of GP cell surface receptors was measured by flow-cytometric analysis and correlated with the GP-specific RNA content. This method may be useful to study the GP-specific RNA content in platelets as well as in other tissues, such as megakaryocytes, especially in patients with congenital or acquired platelet function disorders.