Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.

The scope of this article is to outline some of the basic methods for good quality RNA preparation from mammalian tissues and cells (including epithelial cells). Additionally, we give an outline of common techniques of measuring CFTR gene expression such as quantitative and semi-quantitative reverse transcription (RT) PCR and ribonuclease protection assay (RPA). These methods are designed to detect low abundance transcripts, which apply to CFTR mRNA in most cell types and tissues.

[1]  M. Amaral,et al.  Cystic fibrosis patients with the 3272‐26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane , 1999, Human mutation.

[2]  A. Harris,et al.  Lymphocyte mRNA as a resource for detection of mutations and polymorphisms in the CF gene. , 1991, Journal of medical genetics.

[3]  M. Amaral,et al.  Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis. , 2002, American journal of respiratory cell and molecular biology.

[4]  M. Amaral,et al.  Non-PCR methods for the analysis of CFTR transcripts. , 2004, Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society.

[5]  C. Higgins,et al.  Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes. , 1998, Human molecular genetics.

[6]  M. Tzetis,et al.  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 , 2001, Human Genetics.

[7]  J. Clancy,et al.  Assessment of CFTR localisation in native airway epithelial cells obtained by nasal brushing. , 2004, Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society.

[8]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[9]  L. Tsui,et al.  Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. , 1989, Science.