Cardiac Gene Expression: Methods and Protocols presents both cutting-edge and established methods for studying cardiac gene expression. The protocols provide a template for solid research, and cover the process through screening, analysis, characterization, and functional confirmation of novel genes or known genes with a new function. Section I, Cardiac Gene Expression Profiling: The Global Perspective, discusses several different approaches to examining, identifying, and analyzing changes in transcriptome gene expression. Section II, Cardiac Gene Regulation: Gene-Specific mRNA Measurement in the Myocardium, outlines more sensitive and gene-targeted expression methods. Section III, Cardiac Gene Regulation: Promoter Characterization in the Myocardium, provides protocols for the study of underlying gene regulation mechanisms by focusing on the interaction of transcription factors with their cognate cis binding elements. Section IV, In Silico Assessment of Regulatory cis-Elements and Gene Regulation, and Section V, Cardiac Single Network Polymorphisms, emphasize new analytical approaches for deciphering the functional elements buried in the 3 billion nucleotides of the human genome and other model genomes. The concluding section, Gene Overexpression and Targeting in the Myocardium, highlights methods that facilitate overexpression or cardiac-specific targeted gene deletion.
Table of Contents; Preface; Contributors; PART I. Cardiac gene expression profiling; The global perspective; 1. Microarray analysis of gene expression in murine cardiac graft infiltrating cells; Yurong Liang, Xin Lu, David L. Perkins; 2. Expression Profiling using Affymetrix GeneChip[registered] Probe Arrays; Martina Schinke-Braun, Jennifer A. Couget; 3. Serial analysis of gene expression (SAGE): A useful tool to analyze the cardiac transcriptome; Kirill V. Tarasov, Sheryl A. Brugh, Yelena S. Tarasova, Kenneth R. Boheler; 4. Functional genomics by cDNA subtractive hybridization; Christophe Depre; 5. Statistical methods in cardiac gene expression profiling: from image to function; Sek Won Kong; PART II. Cardiac gene regulation- gene-specific mRNA measurement in the myocardium; 6. Measurement of cardiac gene expression by reverse transcription polymerase chain reaction (RT-PCR); Nicola King; 7. Quantitative (real-time) RT-PCR in cardiovascular research; Kevin John Ashton, John Patrick Headrick; 8. RNase protection assay for quantifying gene expression levels; Yongxia Qu, Mohamed Boutjdir; 9. In situ hybridization, a technique to study localization of cardiac gene expression; Thierry P. Calmels, David Mazurais; PART III. Cardiac gene regulation- promoter characterization in the myocardium; 10. Characterization of cis-regulatory elements and transcription factor binding: Gel mobility shift assay Jim Jung-Ching Lin, Shaun E. Grosskurth, Shannon M. Jaacks, Elisabeth A. Gustafson-Wagner, Qin Wang. 11. Mapping transcriptional start sites and in silico DNA footprinting.; Martin E Cullen, Paul JR Barton; 12. Characterization of cardiac gene promoter activity: reporter constructs and heterologous promoter studies; Hsiao-Huei Chen, Alexandre F. R. Stewart; PART IV. In Silico assessment of regulatory cis-elements and gene regulation; 13. Comparative genomics: A Tool to functionally annotate human DNA; Jan-Fang Cheng, James R. Priest, Len A. Pennacchio; 14. Developing computational resources in cardiac gene expression; Michael B. Bober, Raimond Winslow; PART V. Cardiac single nucleotide polymorphisms; 15. in silico analysis of SNPs and other high throughput data; Neema Jamshidi, Thuy D. Vo, Bernhard O. Palsson; 16. Discovery and identification of sequence polymorphisms and mutations with MALDI-TOF Ms; Dirk van den Boom, Mathias Ehrich; PART VI. Gene overexpression and targeting in the myocardium; 17. Conditional targeting: Inducible deletion by Cre recombinase; Kelly R O'Neal, Ramtin Agah; 18. Cardiomyocyte preparation, culture, and gene transfer; Alexander H. Maass, Massimo Buvoli; 19. Adeno-associated viral vector delivered hypoxia-inducible gene expression in ischemic hearts; Hua Su, Yuet Wai Kan.