vascular disease, molecular biology and gene transfer protocols

[...]... From: Methods in Molecular Medicine, vol 30: Vascular Disease: Molecular Biology and Gene Therapy Protocols Edited by: A H Baker © Humana Press Inc., Totowa, NJ 39 40 Townsend, Newman, and Francis Fig 1 A and B show the efficiency of RNA extraction from human (A) and porcine (B) vascular tissue, per mg starting weight of tissue, using the protocols described here RNA Extraction from Vascular Tissue... exist for various genomes, includFrom: Methods in Molecular Medicine, vol 30: Vascular Disease: Molecular Biology and Gene Therapy Protocols Edited by: A H Baker © Humana Press Inc., Totowa, NJ 25 26 Miano, Garcia, and Krahe ing the human (16,17), and the mouse (18), as well as for a wide variety of other species and model systems for human disease (Research Genetics, Huntsville, AL; http://www.resgen.com)... reaction-single-strand conformation polymorphism (PCR-SSCP) analysis PCR Meth Appl 2, 10–13 16 Nagamine, C M., Chan, K., and Lau, Y-F C (1989) A PCR artifact: Generation of heteroduplexes Am J Hum Genet 45, 337–339 Mapping Human Genes 25 3 Radiation Hybrid (RH) Mapping of Human Smooth Muscle-Restricted Genes Joseph M Miano, Emilio Garcia, and Ralf Krahe 1 Introduction Recent molecular genetic studies in cardiac and. .. 13 Ohshiro, K and Puri, P (1998) Pathogenesis of infantile hypertrophic pyloric stenosis: recent progress Pediatr Surg Int 13, 243–252 14 Gerhard, D S., Kawasaki, E S., Bancroft, F C., and Szabo, P (1981) Localization of a unique gene by direct hybridization in situ Proc Natl Acad Sci USA 78, 3755–3759 15 Copeland, N G and Jenkins, N A (1991) Development and applications of a molecular genetic linkage... studies on gene expression in vascular disease to date have used cultured vascular cells (4–6), as the RNA extraction is easier and the yield greater than from solid tissue However, cell culture per se induces changes in gene expression, and so the use of the intact tissue would be a more valid approach for studying gene expression in vascular disease The successful extraction of RNA from solid vascular. .. distinct band, separated from the corresponding homoduplexes, as their mobility is different There are several detection methods for heteroduplex strands after electrophoresis, but ethidium bromide staining or fluorescence, combined with an automated DNA sequencer, are, in our experience, the best choices From: Methods in Molecular Medicine, vol 30: Vascular Disease: Molecular Biology and Gene Therapy Protocols. .. describe how the combination of PCR and HA can be used as a rapid and simple detection of point mutations in genomic DNA, by means of manual or automated DNA sequencer Main modifications to the HA protocol to carry out CSGE are also described 2 Materials All solutions should be made to the standard required for molecular biology Use molecular biology grade reagents and sterile distilled water 2.1 PCR... Expression, genomic structure and high resolution mapping to 19p13.2 of the human smooth muscle cell calponin gene Gene 197, 215–224 RNA Extraction from Vascular Tissue 39 4 Efficient Extraction of RNA from Vascular Tissue Catherine F Townsend, Christopher M H Newman, and Sheila E Francis 1 Introduction The development of new and effective techniques to study differential gene expression has revolutionized... Human Genes 29 13 10x Tris-Acetate-EDTA: dissolve 48.4 g Tris in deionized water and add 11.4 mL glacial acetic acid and 20 mL 0.5 M EDTA Bring total volume to 1 L and store at room temperature 14 Agarose gels: for a standard 13 × 10 cm gel former, mix 3 g SeaKem LE agarose in 200 mL of 1x TAE, microwave for 2 min and swirl to dissolve 2.2 PCR Amplification of RH Panel 1 RH panel of choice (Research Genetics,... S (1992) Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphisms and doublestranded heteroduplex DNA PCR Meth Appl 2, 96–98 9 Ganguly, A., Rock, M J., and Prockop, D J (1993) Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends . in Molecular Medicine, vol. 30: Vascular Disease: Molecular Biology and Gene Therapy Protocols Edited by: A. H. Baker © Humana Press Inc., Totowa, NJ 2 Analysis of Genetic Variants in Cardiovascular. 3 3 From: Methods in Molecular Medicine, vol. 30: Vascular Disease: Molecular Biology and Gene Therapy Protocols Edited by: A. H. Baker © Humana Press Inc., Totowa, NJ 1 Detection of Mutations and DNA Polymorphisms. described. 2. Materials All solutions should be made to the standard required for molecular biology. Use molecular biology grade reagents and sterile distilled water. 2.1. PCR Reaction for Heteroduplex