Ribonucleic Acid

The current understanding of the important genes and molecular signaling pathways that regulate myocardial remodeling and the transition to heart failure is based largely on animal models and clinical trials. These studies have confirmed the importance of the neurohormonal systems, including the renin-angiotensin-aldosterone axis, the sympathetic nervous system, and natriuretic peptides in the pathogenesis of the heart failure phenotype (2,21-26). Furthermore, a number of recent failed drug trials have highlighted the potential limitations of animal models in replicating a complex disease such as human heart failure.

Collectively, these findings emphasize the need for resources and tools to utilize tissue from the normal and diseased human heart effectively to increase the basic understanding of the molecular determinants governing the transition to heart failure. The advent of high-throughput, genomics-based strategies has provided a leap forward in the ability to accomplish this and thus to discover novel genes and signaling pathways. For example, the present ability to interrogate nearly the entire genome in a single experiment has allowed scientists to develop unconventional and unbiased approaches to solve problems that may provide a fertile environment for novel drug discovery.

A microarray or gene chip contains oligonucleotides (short strings of bases specific for genes) or complementary DNA (cDNA, individual DNA sequences) for hundreds or thousands of genes on a quartz wafer or glass microscope slide (27,28) (Fig. 1). The principle behind this technology is the hybridization potential between nucleic acids. Put simply, this allows a researcher to compare expression of a large number of genes (>22,000) in control and diseased tissues or cells. This is then useful for defining the genes and signaling pathways that may regulate a patient's transition to heart or vascular disease. Furthermore, the identification of these targets can then be used to develop diagnostic tests for early detection and prevention of disease, as well as for novel drug discovery to treat the disease.

Technical use of a microarray depends on the platform employed (oligonucleotide or cDNA). For example, with an oligonucleotide gene chip, the researcher would isolate RNA from the tissue or cells, reverse transcribe the RNA to cDNA, and in vitro transcribe to cRNA with biotin-labeled nucleotides. The biotin-labeled sample is then hybridized to the array, and the arrays are stained with a streptavidin-phycoerythrin conjugate that binds biotin and emits a fluorescent signal. This array is scanned, and the gene expression values are quantitated. For cDNA arrays, RNA from two different tissue or cell populations is isolated and reverse transcribed to cDNA in the presence of nucleotides labeled with two different fluorescent dyes (e.g., Cy3 [green] and Cy5 [red]).

The samples are then simultaneously hybridized to the array, where they "compete" for binding. The slide is scanned, and the fluorescence is quantitated for each spotted cDNA. Finally, sifting and analyzing the wealth of data from microarray experiments is performed with the help of several different statistical and bioinformatics programs (29-32).

A downfall of the microarray approach is the limitation to study only the sequences represented on the developed chip. Although the sensitivity of this approach has improved, the ability to detect genes with low expression levels reproducibly has been a challenge. Fortunately, the establishment of public databases and resources, including those provided by the National Heart, Lung, and Blood Institute's Programs for Genomics Applications (http://www.nhlbi.nih.gov/resources/pga/) and Cardiac Gene Expression (CaGE) Knowledgebase, has been useful in comparing and analyzing gene expression libraries.

Serial analysis of gene expression (SAGE) is a technique that allows definition of the genes in a given tissue or cell type. This approach was developed by Velculescu et al. (33) and is not limited to transcript information printed on a given platform. Briefly, short sequence tags, which carry sufficient information to identify each gene uniquely, are linked together and cloned. Compared to microarrays, SAGE is better able to identify transcripts of low abundance and is thus also perhaps better suited to identify novel genes.

Massively parallel signature sequencing (MPSS) is an emerging technology discovered by the Nobel Prize Laureate Sydney Brenner and colleagues (34). Importantly, this approach is not limited by the sequences spotted on a chip, and its level of sensitivity far outweighs that of the currently used microarray-based platforms. Briefly, MPSS is based on the in vitro cloning of millions of templates on microbeads. Sequencing of the 16-20 base templates on each bead is performed simultaneously using a fluorescence-based signature sequencing approach with repeated cycles of enzymatic cleavage (see ref. 34 for an excellent review). This approach is sensitive as well as high throughput in design.

Microarrays, SAGE, and MPSS have the capability to identify critical genes and regulatory signals governing cardiovascular remodeling and disease as well as possibly identify new clinical biomarkers.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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