Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

AbstractNucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed. FigureThe nucleic acid sequence based amplification (NASBA) reaction taking place on top of a microarray. a: During the NASBA process, biotinylated antisense RNA is produced. b: Hybridization of these amplicons with their immobilized capture probes occurs during amplification. Capture probes are immobilized within spots of a surface-attached hydrogel, arranged in a microarray. Each spot potentially represents a different capture probe. ds cDNA doubled-stranded complementary DNA, PMMA polymethylmethacrylate

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