Abstract
Quantification and analysis of CFTR transcripts is of crucial importance not only for cystic fibrosis (CF) diagnosis and prognosis, but also in evaluating the efficiency of various therapeutic approaches to CF, including gene therapy. Reverse transcription (RT) followed by quantitative polymerase chain reaction (qPCR) is at present the most sensitive method for transcript abundance measurement. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods have evolved towards reliable quantification. In this chapter we describe and discuss several protocols for the quantitative analysis of CFTR transcripts, including those variants that result from alternative splicing.
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References
Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073.
Bremer, S., Hoof, T., Wilke, M., Busche, R., Scholte, B., Riordan, J. R., et al. (1992) Quantitative expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially spliced cystic-fibrosis transmembrane- conductance regulator mRNA transcripts in human epithelia. Eur. J. Biochem. 206, 137–149.
Harris, C. M., Mendes, F., Dragomir, A., Doull, I. J. M., Carvalho-Oliveira, I., Bebok, Z., et al. (2004) Assessment of CFTR localisation in native airway epithelial cells obtained by nasal brushing. J. Cyst. Fibros. 3(Suppl 2), 43–48.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ramalho, A. S., Beck, S., Farinha, C. M., Clarke, L. A., Heda, G. D., Steiner, B., et al. (2004) Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts. J. Cyst. Fibros. 3(Suppl 2), 11–15.
Ramalho, A. S., Beck, S., Meyer, M., Penque, D., Cutting, G. R., and Amaral, M. D. (2002) Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 27, 619–627.
Clarke, L. A., and Amaral, M. D. (2004) Semi-Quantitative RT-PCR Analysis of CFTR mRNA Abundance. European working group on CFTR expression, virtual repository. http://central.igc.gulbenkian.pt/cftr/vr/a/clarke_semi_quantitative_rt_pcr_analysis_cftr_mrna.pdf
Chasin, L. A. (2007) Searching for splicing motifs. Adv. Exp. Med. Biol. 623, 85–106.
Pagani, F., Buratti, E., Stuani, C., and Barelle, F. E. (2003) Missense, nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9. J. Biol. Chem. 278, 26580–26588.
Ramalho, A. S., Beck, S., Penque, D., Gonska, T., Seydewitz, H. H., Mall, M., et al. (2003) Transcript analysis of the cystic fibrosis splicing mutation 1525-1G>A shows use of multiple alternative splicing sites and suggests a putative role of exonic splicing enhancers. J. Med. Genet. 40, e88.
Tzetis, M., Efthymiadou, A., Doudounakis, S., and Kanavakis, E. (2001) Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A→G, 2751+2T→A, 296+1G→C, 1717-9T→C-D565G) and one nonsense mutation (E822X) in the CFTR gene. Hum. Genet. 109, 592–601.
Dork, T., Fislage, R., Neumann, T., Wulf, B., and Tümmler, B. (1994) Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations. Hum. Genet. 93, 67–73.
Zink, D., Amaral, M. D., Englmann, A., Lang, S., Clarke, L. A., Rudolph, C., et al. (2004) Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei. J. Cell. Biol. 166, 815–825.
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Ramalho, A.S., Clarke, L.A., Amaral, M.D. (2011). Quantification of CFTR Transcripts. In: Amaral, M., Kunzelmann, K. (eds) Cystic Fibrosis. Methods in Molecular Biology, vol 741. Humana Press. https://doi.org/10.1007/978-1-61779-117-8_9
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DOI: https://doi.org/10.1007/978-1-61779-117-8_9
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