The construction of gene expression databases requires technologies that can accurately and reproducibly measure changes in global mRNA expression levels. Ideally, these technologies should be able to screen all gene transcripts, be applicable across a wide range of cell and tissue types, require minimal amounts of biological material, and be capable of processing large number of samples. Although several different technology platforms have been developed [4-9], each comes with its own set of advantages and limitations in meeting these stringent requirements (Fig. 1.2). The two most commonly used platforms are complementary DNA (cDNA) microarrays and high-density oligonucleotide arrays.
Proteomic and Genomic Analysis of Cardiovascular Disease. Edited by Jennifer E. van Eyk, Michael J. Dunn
Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30596-3
Total RNA or PolyA4-
Cy3 or Cy5 labeled cDNA
First-strand cDNA synthesis
First-strand cDNA synthesis
I Raw Data Output I
Signaling Ratio of Cy3/Cy5
Normalization t Statistical tests
Clustering algorithms iff
Total RNA or PolyA*
cDNA synthesis i
I in vitro transcription |
| Hybridization and Staining |
Normalization t Statistical tests
Avg Diff / Signal
Clustering algorithms cDNA microarrays have been developed by several academic groups as well as commercial suppliers. In general, cDNA libraries or clone collections are amplified by polymerase chain reaction (PCR) at an average product size of 1,000 nucleotides in length. These PCR products are printed in a two-dimensional grid onto glass slides or nylon membranes as spots at defined locations. Spots are typically 100-300 ^m in size and are spaced about the same distance apart. Using this technique, arrays consisting of more than 30,000 cDNAs can be fitted onto the surface of a conventional microscope slide (Fig. 1.2).
The process of spotting is generally not accurate enough to allow direct comparison between different arrays. Therefore, the two RNA samples from tissues to be compared are typically used to generate first-strand cDNA targets labeled with two different fluorescent dyes, for example Cy3 and Cy5. These are then purified, pooled and hybridized to the same array, resulting in competitive binding of the differentially labeled cDNAs to the arrayed sequences (Fig. 1.1). After hybridization and washing, the slide is scanned in a high-resolution confocal fluorescent scanner using two different wavelengths corresponding to the dyes used, and the intensity of the same spot in both channels is compared. From these measurements, a ratio of transcript levels for each gene represented on the array can be calculated. In order to be able to compare results from different arrays to each other, a reference RNA, e.g. a mixture of all the samples of one experiment or a commercially available standard, is used to normalize the Cy3 and Cy5 intensities (see 2.2 Comparing expression data).
Protocols for total RNA isolation and nucleic acid labeling are available on the internet from a variety of academic laboratories (Tab. 1.1). To achieve the most linear relationship between starting material and labeled probe, incorporation of fluorescent-labeled nucleotides during first strand cDNA synthesis has been the method of choice. However, this requires up to 100 ^g of total RNA as starting material, which excludes studies using primary cells or human tissues of limited availability.
Fig. 1.1 Schematic overview of cDNA and oligonucleotide microarray sample preparation. Total RNA is isolated from tissues of the experimental sample (e.g. hypertrophic heart), and from the control sample (e.g. normal heart). A For cDNA microarrays, RNA from both samples is reverse transcribed into single-stranded cDNA in the presence of two fluorescent dyes (such as Cy3 and Cy5). Both samples are mixed in the hybridization buffer and hybridized to the array, usually under a coverslip. After washing, array slides are scanned by a high-resolution confocal fluorescence scanner with two wavelengths corresponding to the two fluorescent dyes used, and independent images for the control and experimental channels are generated. Signal intensities for both dyes are used to calculate the ratios of mRNA abundance. B Total RNA is isolated from the experimental and control tissue sample, and cDNA is synthesized using an Oligo-dT primer that carries the T7 promotor sequence. T7 DNA polymerase is then used for in vitro transcription with biotin-labeled nucleotides that become incorporated into the cRNA. Each labeled cRNA is then hybridized to an array in a rotating hybridization oven. After hybridization, the array is washed and stained using a fluorescent dye coupled to streptavidin. Arrays are scanned and signal intensities are converted into Average Difference (AvgDiff) values or Signals. Modified from .
Tab. 1.1 Links to Microarray Resources. This table provides a selection of commercial and academic facilities and resources related to microarray technologies
Microarray Core Facilities
Public Microarray Databases http://pga.lbl.gov/PGA/PGA_inventory.html
Protocols and Software
Stanford DNA Sequencing and Technology Center
Bauer Center for Genomics Research National Human Genome Research Institute
Microarrays Inc., Nashville, TN
NHLBI Programs for Genomic Applications Inventory Site
Raw data and normalized data related to cardiomyopathies in mouse models and humans using Affymetrix GeneChipsTM Microarray data storage, image files, data retrieval, analysis, and visualization Gene expression and hybridization array data repository, and online resource for the retrieval of gene expression data from any organism or artificial source Public database for microarray based gene expression data
Affymetrix online resource center for GeneChip arrays dCHIP, freely available analysis tool for Affymetrix GeneChip arrays A guide to cDNA microarrays
Public source for microarray protocols and software
Software tools freely available to the scientific community
Software tools for image analysis, and cluster analysis and visualization Commercial software products for mi-croarray research and analysis Set of tools for clustering, analysis and visualization of gene expression data
Development of standards for array experiment annotation and data representation
http://www.mged.org Microarray Gene Expression Database
cDNA collection or libraries
Insert a mpl ifi c ati on by PCR I
F't I : t r I j ! Spotting Coupling Denaturing cDNA spotted arrays
1.1 DNA Microarray Technologies | 7 g rnRNA Reference Sequence j Probe Set \
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