Simultaneous determination of multiple mRNA levels utilizing MALDI-TOF mass spectrometry and biotinylated...

Here we report an efficient method to simultaneously measure multiple mRNA levels utilizing mass spectrometry (MS) and molecular affinity isolation. In this approach, reverse transcription products of a group of mRNAs are subjected to competitive PCR with competitors and internal standards of known concentrations, and the PCR products are differentiated and quantified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS to determine the mRNA levels. The method provides high accuracy in quantitative MS analysis due to the facilitated purification of oligonucleotides by molecular affinity isolation. Additionally, owing to the molecular affinity isolation, only those oligonucleotides required for expression level determination are introduced into the mass spectrometer, while other irrelevant reaction components that could overlap with peaks of gene transcripts or competitors are removed prior to MS analysis. Thus the approach enhances the parallel analysis of multiple gene transcripts by MS. Utilizing the method we have simultaneously measured mRNA levels of four genes (Rho, Nrl, Hprt, and Lhx2) in mouse retinal tissue.