gms | German Medical Science

Objective: TaqMan^® Protein Assays is an innovative method, which detects proteins through amplification of substitute DNA templates. Therefore TaqMan^® Protein Assays use antibodies and proximity ligation for quantitative Real-Time PCR. In this study we evaluated TaqMan^® Protein Assays in different human tissues, cancer cell lines and meningiomas.

Method: RNA and proteins from five random meningiomas and the human glioblastoma cell line U373 MG were co-isolated and quantified. RNA and proteins of eight different human tissues (Brain, Cerebral Meninges, Colon, Kidney, Liver, Lung, Pancreas and Placenta) and seven cancer cell lines (A-431, Hl-60, HeLa, HepG2, Jurkat, K-562 and MCF-7) were purchased. Five angiogenesis proteins were determined: VEGFA, VEGFR-2 (KDR), Neuropilin 1 (NRP1), PDGF-B and PDGFR-β. For quantitative Real-Time PCR TaqMan^® Gene Expression Assays were used. For TaqMan^® Protein Assays biotinylated full length antibodies were labeled with oligonucleotides. Detection limit was compared with Western Blot analysis.

Results: Human brain was used for relative quantification in quantitative Real-Time PCR and TaqMan^® Protein Assays. PDGFR-β and KDR expression was not detected in almost all probes (2 of 21) with Western Blot analysis. With TaqMan^® Protein Assays PDGFR-β and KDR expression was detected in 18 of 21 protein lysates. VEGFA, PDGF-BB and NRP1 were detected in all probes with TaqMan^® Protein Assays, but not in Western Blots. There were differences between RNA and protein levels both with increased protein and RNA level.

Conclusions: TaqMan^® Protein Assays allowed the detection and quantification of very low protein expression levels in human tissues, cancer cell lines and meningioma primary cells. Direct correlation of relative RNA quantities and associated protein levels showed in part distinct differences.