Real-time PCR and its application for protein quantification

Doctoral thesis, 2007

The ability to quantify specific DNA sequences with real-time PCR has increased the possibilities for sensitive DNA analysis enormously. This sensitive technique can in optimal conditions detect one DNA copy and has a large quantification range. The method is based on exponential amplification of a DNA sequence that is detected by a fluorescent signal during the reaction. The fluorescence comes either from a sequence specific probe or a nonspecific DNA binding dye. This thesis describes a new way to use sequence specific probes and DNA binding dye in combination to get more information from the reaction. The thesis also describes the development of immuno-qPCR, which is a sandwich immunoassay that uses real-time PCR for sensitive detection and quantification of proteins. A DNA-label on the detection antibody is used for signal amplification through real-time PCR. The amount of DNA-label will correspond to the amount of the protein analyte which then indirectly is quantified. Assays for sensitive quantification of two different proteins, prostate specific antigen (PSA) and protein A (which also is a part of the tandem affinity purification (TAP)-tag) was developed and applied to different areas. Three ways to assemble the assay for PSA quantification was compared. The sensitivity, reproducibility and ease of use were evaluated. It was found that a covalent conjugate between antibody and DNA-label was important to get as high reproducibility and sensitivity as possible by reducing the handling steps during assay performance. A sensitivity increase of more than 100 times compared to ELISA was achieved. The PSA assay was used for quantification of PSA protein levels in laser microdissected prostate tissue. This was done in combination with quantification of PSA mRNA transcripts, by real-time RT-PCR, from corresponding tissue samples. The immuno-qPCR assay for quantification of the TAP-tag was shown to work well for quantification of TAP-tagged proteins in Saccharomyces cerevisiae. The TAP-protein was also used as a reporter gene to study the effect of different 3’ untranslated regions (UTR). Measurements were done on both protein and mRNA level and showed, indeed, that the 3’UTR contains important regulatory elements and can change the expression several fold.