Multiplexed gene expression analysis using the invader RNA assay with MALDI-TOF mass spectrometry detection.
A mass spectrometric approach for measuring gene expression levels has been developed. This 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.
Berggren
W Travis
WT
Department of Chemistry, University of Wisconsin at Madison, 53706, USA. wtberggr@students.wisc.edu
Takova
Tsetska
T
Olson
Marilyn C
MC
Eis
Peggy S
PS
Kwiatkowski
Robert W
RW
Smith
Lloyd M
LM
D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D015347:RNA Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D007375:Interleukin-1; D015850:Interleukin-6; D015347:RNA Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases