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Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay.
RNA. 2003 Dec; 9(12):1552-61.RNA

Abstract

The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing.

Authors+Show Affiliations

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14624010

Citation

Wagner, Eric J., et al. "Quantification of Alternatively Spliced FGFR2 RNAs Using the RNA Invasive Cleavage Assay." RNA (New York, N.Y.), vol. 9, no. 12, 2003, pp. 1552-61.
Wagner EJ, Curtis ML, Robson ND, et al. Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay. RNA. 2003;9(12):1552-61.
Wagner, E. J., Curtis, M. L., Robson, N. D., Baraniak, A. P., Eis, P. S., & Garcia-Blanco, M. A. (2003). Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay. RNA (New York, N.Y.), 9(12), 1552-61.
Wagner EJ, et al. Quantification of Alternatively Spliced FGFR2 RNAs Using the RNA Invasive Cleavage Assay. RNA. 2003;9(12):1552-61. PubMed PMID: 14624010.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay. AU - Wagner,Eric J, AU - Curtis,Michelle L, AU - Robson,Nicole D, AU - Baraniak,Andrew P, AU - Eis,Peggy S, AU - Garcia-Blanco,Mariano A, PY - 2003/11/19/pubmed PY - 2004/1/17/medline PY - 2003/11/19/entrez SP - 1552 EP - 61 JF - RNA (New York, N.Y.) JO - RNA VL - 9 IS - 12 N2 - The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/14624010/Quantification_of_alternatively_spliced_FGFR2_RNAs_using_the_RNA_invasive_cleavage_assay_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=14624010 DB - PRIME DP - Unbound Medicine ER -