Quantitative transcriptional profiling with the aid of affinity capture

We have developed a novel method for a truly quantitative analysis of transcriptional profiles in cell samples. This low cost method is based on liquid sandwich hybridization between mRNA molecules, fluorophore labelled DNA-fragments of given sizes as probes and biotinylated oligo(dT). The mRNA : probe : biotin-oligo(dT) -complexes are separated in an automated fashion using magnetic (strept)avidin-coated microparticles. After washes the probes are eluted and their identity and quantity determined by electrophoretic analysis on an automated sequencer or by MS analysis.This method, which can be used also for uncharacterised genomes, gives the data as the copynumber of each identified mRNA. As a practical performance the process is simple and can be performed to a great part with standard laboratory equipments. The labelled DNA-fragment library is built using computer aided planning and PCR and allows simple assembly of custom made gene probe sets. This feature makes this method an ideal alternative to expensive microarrays and high density oligonucleotide chips e.g. in clinical settings, where the focus of the analysis is among a subset of genes.