VTT-TRAC for focused transcript analysis

DNA microarrays allowing genome-wide transcriptional profiling have become key tools in research. Accumulation of transcriptional profiling data and defined molecular signatures have lead to an increasing demand for focused arrays to reduce costs and bioinformatics overload in routine applications. We have developed an alternative method VTT-TRAC (transcript analysis with the aid of affinity capture) for multiplexed and quantitative analysis of transcripts (Ref). It is based on solution hybridization between RNA and multiple fluorophore labeled probes of distinct sizes. The RNA-probe complexes are captured on streptavidin-coated magnetic beads. For this purpose, RNA has been previously biotinylated chemically or biotin-oligo-dT capture probe is used to mediate the capture. After washes, hybridized probes are eluted and analyzed by capillary electrophoresis. The probe signal intensities correspond to the amount of RNA, while the probe size indicates the target. We have successfully applied VTT-TRAC in several applications: transcript analysis of eukaryotic microbes S.cerevisiae and T.reesei and quantification of different bacterial groups in a mixed population. Currently we are developing an assay for expression analysis of colon cancer related genes. We have used probes of variable size from oligonucleotides to few hundred bp. While oligonucleotides enable fast assays eg for bacterial detection and bioprocess monitoring, longer probes with universal linkers enable ultra-sensitive detection. Long probes may be amplified after elution by using the universal linkers as primer sites. The ultra-sensitive assay could prove valuable eg in cancer diagnostics from clinical tissue samples with a limited amount of cells. VTT-TRAC provides a practical alternative for focused transcript analysis. lt is sensitive, accurate and convenient multiplex method, which does not necessitate mRNA conversion to cDNA. Reference: Söderlund et al. Finnish pat. FI20010041 / Int.pat.appl. No. PCT/FIO2/00023