Author/Editor		Ficko, Tanja; Černelč, Peter
Title		Real-time quantitative PCR assay for analysis of platelet glycoprotein IIIa gene expression
Type		članek
Source		J Biochem Biophys Methods
Vol. and No.		Letnik 62, št. 3
Publication year		2005
Volume		str. 241-50
Language		eng
Abstract		A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.
Descriptors		ANTIGENS, HUMAN PLATELET BASE SEQUENCE BLOOD PLATELETS DNA, COMPLEMENTARY POLYMERASE CHAIN REACTION RNA, RIBOSOMAL, 18S REPRODUCIBILITY OF RESULTS SENSITIVITY AND SPECIFICITY TIME FACTORS