High-throughput multiplexed transcript analysis yields enhanced resolution of 5-hydroxytryptamine2C receptor mRNA editing profiles

Michael V. Morabito, Randi J. Ulbricht, Richard T. O'Neil, David C. Airey, Pengcheng Lu, Bing Zhang, Lily Wang, Ronald B. Emeson

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-toinosine editing). Commonly used 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 more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine2C; 5HT2C) transcript, an RNA editing substrate in which 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 the 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 5HT 2C 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 pharmacological and physiological perturbation, further elucidating the impact of 5HT2C RNA editing on central nervous system function.

Original languageEnglish (US)
Pages (from-to)895-902
Number of pages8
JournalMolecular Pharmacology
Volume77
Issue number6
DOIs
StatePublished - Jun 1 2010
Externally publishedYes

Fingerprint

RNA Editing
Messenger RNA
Adenosine
Complementary DNA
Clone Cells
Receptor, Serotonin, 5-HT2C
Inosine
Inbred Strains Mice
Selection Bias
Sequence Analysis
Protein Isoforms
Central Nervous System
Pharmacology
Brain

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Morabito, M. V., Ulbricht, R. J., O'Neil, R. T., Airey, D. C., Lu, P., Zhang, B., ... Emeson, R. B. (2010). High-throughput multiplexed transcript analysis yields enhanced resolution of 5-hydroxytryptamine2C receptor mRNA editing profiles. Molecular Pharmacology, 77(6), 895-902. https://doi.org/10.1124/mol.109.061903

High-throughput multiplexed transcript analysis yields enhanced resolution of 5-hydroxytryptamine2C receptor mRNA editing profiles. / Morabito, Michael V.; Ulbricht, Randi J.; O'Neil, Richard T.; Airey, David C.; Lu, Pengcheng; Zhang, Bing; Wang, Lily; Emeson, Ronald B.

In: Molecular Pharmacology, Vol. 77, No. 6, 01.06.2010, p. 895-902.

Research output: Contribution to journalArticle

Morabito, Michael V. ; Ulbricht, Randi J. ; O'Neil, Richard T. ; Airey, David C. ; Lu, Pengcheng ; Zhang, Bing ; Wang, Lily ; Emeson, Ronald B. / High-throughput multiplexed transcript analysis yields enhanced resolution of 5-hydroxytryptamine2C receptor mRNA editing profiles. In: Molecular Pharmacology. 2010 ; Vol. 77, No. 6. pp. 895-902.
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