RT Journal Article SR Electronic T1 High-throughput multiplexed transcript analysis yields enhanced resolution of 5HT2C receptor mRNA editing profiles JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.109.061903 DO 10.1124/mol.109.061903 A1 Michael V Morabito A1 Randi J Ulbricht A1 Richard T O'Neil A1 David C Airey A1 Pengcheng Lu A1 Bing Zhang A1 Lily Wang A1 Ronald B Emeson YR 2010 UL http://molpharm.aspetjournals.org/content/early/2010/02/24/mol.109.061903.abstract AB RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (A-to-I editing). Commonly employed methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of over 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5HT2C) transcript, an RNA editing substrate where up to five adenosines are modified. Using a high-throughput multiplexed transcript analysis we were able to quantify accurately the expression of twenty 5HT2C isoforms, each representing at least 0.25% of total 5HT2C transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT2C editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT2C variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacologic and physiologic perturbation, further elucidating the impact of 5HT2C RNA editing on central nervous system function.The American Society for Pharmacology and Experimental Therapeutics