Abstract
Abstract Gene expression analysis in the oncology space is largely performed with the classic and widely accepted methods real-time RT-PCR, microarray and next generation sequencing. These technologies are, however, too time consuming and expensive to be used in large scale screens with high sample numbers. TRAC (Transcription analysis with the aid of Affinity Capture) technology provides a solution for focused gene expression screening with unique combination of multiplex analysis, high-throughput sample processing, high precision and flexibility. Lysates of cells or tissues are directly applicable as sample material in TRAC assay without RNA extraction or cDNA conversion. The assay enables detection of up to 30 targets per sample and 96 samples per assay with 3-4h run time. In TRAC assay, each target mRNA of interest is recognized by a specific labeled ssDNA probe. Probes with different lengths and labels can be used for high-level of multiplexing. Hybridization of probes and targets takes place in solution, after which the probe-transcript complexes are captured by streptavidin coated magnetic beads. Unbound material is washed off and the probes are eluted for detection. The probes are then detected and quantified by capillary electrophoresis, which resolves the probes according to both size and fluorescence. The aim of this study was to apply TRAC assay to analyze the expression fingerprints of 20 gene markers related to angiogenesis, cell adhesion, protease activity and plasminogen activation, in cultured cells treated with different drug candidates. The expression of the target genes was analyzed from four different colon cancer cell lines (COLO, HT-29, CaCo2, DLD) to study culture age dependent effects and responses to drug candidates. Out of the 20 genes analyzed, curcumin caused consistent variation to cyclooxygenase-1 (COX-1) gene expression as a function of time in all tested cell lines. By comparison of expression fingerprints between non-treated cell line cultures, altogether four genes were identified as being expressed at higher level in one or two of the cell lines compared to the others. These genes were trypsinogen (PRSS1-3) in COLO, Serine protease inhibitor (SPINK) in HT-29, Protease Cathepsin B (CTSB) in COLO and cyclin D1 (CCDN1) in DLD and CaCo2 cell lines. The data showed good correlation with real-time RT-PCR results and reproducibility (CV <12%) and functioned as proof-of-concept for suitability of TRAC for screening purposes. Multiplex detection with TRAC from lysates offered high efficiency and significant cost and time savings compared to single-plex qPCR assay in large scale evaluation of drug candidates. Citation Format: Laura Mattinen, Jani Salmivaara, Jussi Halleen, Hans Söderlund, Jari Rautio. TRAC, a novel time and cost saving gene expression analysis technology with improved efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3610.
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