(283c) Simultaneous Detection of Multiple miRNAs for Epidemiologic Study of Alcoholism

MicroRNAs (miRNAs) are thought to regulate mRNA stability and, with their target genes, constitute coordinated genetic networks important for modulating behavioral adaptations. For instance, miRNA-gene networks contribute to the epidemiologic diversity in alcohol drinking. People differ in their propensity both for alcohol preference and for relapse to alcohol drinking, and it is well-known that genetic factors contribute to this epidemiologic diversity in alcohol drinking and alcoholism. The specific genetic factors that predispose recovering alcoholics to relapse are not well-understood.

Most common methods for investigating miRNAs and their target genes include microarray-based methods and RT-PCR. Although these methods have been widely used in many transcriptional assays, they have drawbacks such as requirement of large sample amount (microarray) and low throughput (RT-PCR). Therefore, for genetic studies of epidemiologic diversity in alcohol drinking behavior, we have developed a method involving matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).

Our method allows fast and accurate analysis of multiple miRNAs and mRNAs, with high efficiency and reliability. It employs SPC-SBE, a robust genotyping approach that uses molecular affinity between biotin and biotin-binding protein such as streptavidin and avidin, for highly multiplexed and quantitative analysis of gene transcripts. Briefly, reverse transcription (RT) products and known amount of internal standards (competitors), one for each selected transcript are PCR-amplified competitively in one reaction tube. After the competitive PCR reaction, a library of single base extension (SBE) primers of a particular length and structure is added for extension at the variation site using biotin-ddNTPs in one reaction tube. Subsequently, extension products are isolated by solid phase capture and analyzed with MALDI-TOF MS. The ratio of mass spectral peak areas is measured to determine the relative quantity of the transcripts and their competitors.

In this study, we used the method to investigate seven miRNAs that correlate closely with alcohol preference together with a set of genes whose mRNAs are potential targets of these miRNAs in a mouse model. As shown in this study, our new approach is crucial for exploration of potential miRNA-gene networks involved in the epidemiologic diversity.