CDNA-based microarrays, 185

Cristillo AD, Bierer BE. Identification of novel targets of immunosuppressive agents by cDNA-based microarray analysis. J Biol Chem 2002 277 4465-76. 

Slide-based microarray technology was first introduced by Schena etal. (1995). The processes and equipment for preparing (arrayer) and analyzing (laser scanner) microarray slides comprised a portion of Dari Shalon s thesis work at Stanford University. Polymerase chain reaction (PCR) products (cDNA probes) were attached to PLL-coated glass microscope slides. The... 

Fig. 1. A high-throughput platform of the carbohydrate-based microarrays. A high-precision robot designed to produce cDNA microarrays was utilized to spot carbohydrate antigens onto a chemically modified glass slide. The microspotting capacity of this system is approximately 20,000 spots per chip. The antibody-stained slides were then scanned for fluorescent signals with a Biochip Scanner that was developed for cDNA microarrays. The microarray results were subsequently confirmed by at least one of the conventional alternative assays.

The basis of contemporary microarray technology is printing known oligonucleotide sequences in predefined positions onto the array surface, which then capture the labeled complementary strands from the sample, following the complementarity rules (duplex forming and hybridization). After hybridization, the original amount of mRNA molecules can be estimated from the signal intensity of the captured/hybridized molecules. In the case of the microscope slide-based microarrays (also referred to as cDNA microarray), presynthesized complementary DNA molecules are attached to a solid surface [40]. 

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

Katsuma, S., Nishi, K., Tanigawara, K., Ikawa, H., Shiojima, S., Takagaki, K., Kamin-ishi, Y., Suzuki, Y., Hirasawa, A., Ohgi, T., Yano, J., Murakami, Y., and Tsujim-oto, G., Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling, Biochem. Biophys. Res. Commun., 288, 747-751, 2001. 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

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