Quantification of RNA editing of the serotonin 2C receptor (5-HT2CR) ex vivo

Maria Fe Lanfranco, Noelle Anastasio, Patricia K. Seitz, Kathryn Cunningham

Research output: Book/ReportBook

1 Citation (Scopus)

Abstract

The 5-HT2CR receptor (5-HT2CR) exerts tonic and phasic inhibitory influence over brain circuitry, and dysregulation of this influence contributes to the neurochemical underpinnings in the etiology of a variety of neuropsychiatric disorders including addiction, depression, and schizophrenia. A strategically important regulator of the 5-HT2CR function and protein diversity is mRNA editing, a type of posttranscriptional modification that alters codon identity and thus the translation of distinct, though closely related, isoforms of 5-HT2CR from a single, original transcript. The 5-HT2CR mRNA can be edited at five closely spaced sites, altering the identity of up to three amino acids in the predicted second intracellular loop of the receptor to modulate receptor:G-protein coupling and constitutive activity. Methods to study changes in mRNA editing based upon direct DNA sequencing are both time and labor intensive. To streamline the acquisition of mRNA editing data and improve quantification, we have adapted real-time reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantify mRNA editing in 5-HT2CR transcripts by utilizing TaqMan® probes modified with a minor groove binder (MGB). The method is very sensitive, detecting as little as 10-18g (1attogram) of standard cDNA template and can discriminate closely related 5-HT2CR mRNA edited isoforms. This technique expands the breadth of available quantification methods for mRNA editing and is particularly useful for the ex vivo analyses of mRNA editing of the 5-HT2CR by allowing the rapid collection of data on large numbers of tissue samples. In addition, the general technique can be adapted easily to investigate edited mRNA from other genes, thus facilitating the development of a broader knowledge base of the physiological role of mRNA editing.

Original languageEnglish (US)
PublisherUnknown Publisher
Number of pages18
Volume485
EditionC
DOIs
StatePublished - 2010

Publication series

NameMethods in Enzymology
No.C
Volume485
ISSN (Print)00766879

Fingerprint

Receptor, Serotonin, 5-HT2C
RNA Editing
RNA
Messenger RNA
RNA Isoforms
Polymerase Chain Reaction
Knowledge Bases
Polymerase chain reaction
Transcription
DNA Sequence Analysis
GTP-Binding Proteins
Codon
Reverse Transcription
Binders
Brain
Schizophrenia
Protein Isoforms
Complementary DNA
Genes
Personnel

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Lanfranco, M. F., Anastasio, N., Seitz, P. K., & Cunningham, K. (2010). Quantification of RNA editing of the serotonin 2C receptor (5-HT2CR) ex vivo. (C ed.) (Methods in Enzymology; Vol. 485, No. C). Unknown Publisher. https://doi.org/10.1016/B978-0-12-381296-4.00018-X

Quantification of RNA editing of the serotonin 2C receptor (5-HT2CR) ex vivo. / Lanfranco, Maria Fe; Anastasio, Noelle; Seitz, Patricia K.; Cunningham, Kathryn.

C ed. Unknown Publisher, 2010. 18 p. (Methods in Enzymology; Vol. 485, No. C).

Research output: Book/ReportBook

Lanfranco, MF, Anastasio, N, Seitz, PK & Cunningham, K 2010, Quantification of RNA editing of the serotonin 2C receptor (5-HT2CR) ex vivo. Methods in Enzymology, no. C, vol. 485, vol. 485, C edn, Unknown Publisher. https://doi.org/10.1016/B978-0-12-381296-4.00018-X
Lanfranco, Maria Fe ; Anastasio, Noelle ; Seitz, Patricia K. ; Cunningham, Kathryn. / Quantification of RNA editing of the serotonin 2C receptor (5-HT2CR) ex vivo. C ed. Unknown Publisher, 2010. 18 p. (Methods in Enzymology; C).
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