A mass spectrometric approach for measuring gene expression levels has been developed. Ibis technique utilizes a signal amplification system and analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Signal amplification from the targeted RNA employs a recently developed invasive cleavage assay that does not require prior PCR amplification. The assay uses a set of target-specific probes (oligonucleotides), which hybridize to the RNA being measured to create an overlap structure with a single-stranded flap. This flap is enzymatically cleaved and accumulates linearly in a target-specific manner. The products of the reaction, short DNA oligomers, are well suited for quantitative detection by MALDI-TOF mass spectrometry. Multiplexing is achieved by designing the assays so that reaction products for different mRNA targets have discrete masses that can be resolved in a single mass spectrum. Simultaneous analysis of human cytokine in vitro transcripts IL-1beta, TNF-alpha, and IL-6, with GAPDH as a reference standard, was used as a model system to demonstrate this novel method of gene expression analysis.
