(536d) Quantification of Multiple Microrna Levels Using Molecular Affinity Isolation and Mass Spectrometry

MicroRNAs (miRNAs) are ~22 nt endogenous RNAs that generally down-regulate gene transcript levels by binding to a messenger RNA (mRNA) with partial base complimentarity. MiRNA, like mRNA, have been implicated in a wide variety of diseases. Here we present a novel method for the quantification of microRNA levels from mammalian tissues. MALDI-TOF MS has been extensively studied for DNA analysis, including DNA sequencing, SNP genotyping, allele frequency determination, and gene transcript measurement since it is accurate, suitable for quantification and easy for automation. Previously, Solid Phase Capturable Single Base Extensions (SPC-SBE), a multiplex genotyping method using biotinylated dideoxynucleotides (biotin-ddNTPs) and single base extension reactions, has been developed for MALDI-TOF based genetic variation detection and for gene transcript quantification. The method involves generating a library of oligonucleotide primers that are each extended by a single base corresponding to the site of a polymorphism using biotin-ddNTPs. The DNA extension fragments containing biotin moiety at their 3' end are readily isolated from reaction mixture by utilizing streptavidin-coated microbeads. The isolated DNA molecules are then analyzed with MALDI-TOF MS, allowing accurate DNA analysis and quantification. We are exploring the use of biotin-ddNTPs and MALDI-TOF MS for miRNA expression analysis. This approach for effective miRNA analysis, combined with previously established methods for rapidly examining multiple gene transcript concentrations in one assay, will be highly useful for investigation of gene expression networks in various systems biology studies. Briefly, cDNAs will be reversely transcribed from miRNAs isolated from mouse brain tissue samples and the cDNAs of interest and a known amount of internal standard will be PCR-amplified. A library of primers, one for each cDNA amplicon, will be extended using biotin-ddNTPs alongside the amplified internal standards and cDNAs. Extension products will be isolated by SPC on streptavidin-coated microbeads and analyzed with MALDI-TOF. Ratios between mass spectral peak areas will be used to determine the relative quantity of each transcript. The proposed system will be able to accurately and quickly quantify the expression levels of multiple miRNAs simultaneously and offers several advantages over previously established methods. MALDI-TOF MS is an ideal platform for rapid quantification, and SPC-SBE eliminates artifacts from prior experimental stages to ensure high quality spectra ideal for quantification purposes.