Quantification of CFTR Transcripts

Methods in Molecular Biology

Volumn 741, Issue , 2011, Pages 115-135

  Ramalho, Anabela S ^a,b   Clarke, Luka A ^a   Amaral, Margarida D ^a,b,c  

^a UNIVERSITY OF LISBON   (Portugal)

^b NATIONAL INSTITUTE OF HEALTH DR RICARDO JORGE   (Portugal)

^c EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

Author keywords

CFTR; Cystic fibrosis; internal standards; mRNA; primers; quantification; real time PCR; RT PCR; splicing variants; transcripts

Indexed keywords

ACTIN; CELL EXTRACT; COMPLEMENTARY DNA; ISOPROTEIN; MESSENGER RNA; PRIMER DNA; TRANSMEMBRANE CONDUCTANCE REGULATOR; TRYPSIN;

AGAR GEL ELECTROPHORESIS; ALLELE; ARTICLE; BRONCHUS; CALIBRATION; CELL CULTURE; CYSTIC FIBROSIS; CYTOLOGY; FRACTIONATION; GENETIC PROCEDURES; GENETICS; HUMAN; ISOLATION AND PURIFICATION; METABOLISM; NOSE MUCOSA; NUCLEOTIDE SEQUENCE; PATHOLOGY; QUALITY CONTROL; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; SPECTROPHOTOMETRY; STANDARD;

ACTINS; ALLELES; BASE SEQUENCE; BRONCHI; CALIBRATION; CELL EXTRACTS; CELLS, CULTURED; CHEMICAL FRACTIONATION; CYSTIC FIBROSIS; CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; DNA PRIMERS; DNA, COMPLEMENTARY; ELECTROPHORESIS, AGAR GEL; GENETIC TECHNIQUES; HUMANS; NASAL MUCOSA; PROTEIN ISOFORMS; QUALITY CONTROL; REFERENCE STANDARDS; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; RNA, MESSENGER; SPECTROPHOTOMETRY; TRYPSIN;

EID: 80052341883     PISSN: 10643745     EISSN: 19406029     Source Type: Book Series    
DOI: 10.1007/978-1-61779-117-8_9     Document Type: Chapter

References (13)

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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
8. Chasin, L. A. (2007) Searching for splicing motifs. Adv. Exp. Med. Biol. 623, 85–106.
9. Pagani, F., Buratti, E., Stuani, C., and Barelle, F. E. (2003) Missense, non- sense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9. J. Biol. Chem. 278, 26580–26588.
10. 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.
11. 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.
12. 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.
13. 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.