Application of functional genomics in allergy and clinical immunology

摘要

Although microarray as a research tool seems to be new, the idea of detection of multiple gene expression during one experiment has been present for many years, perhaps starting with reprobing Northern blots with various probes. More recently, RNase protection assay with multiple probes, differential display and serial analysis of gene expression (SAGE) (1) were used. These techniques, although laborious, allowed expression detection of up to 20 or so genes in one RNA sample. The current response to this demand was cDNA microarray developed by Patrick Brown and his group in 1994 (2, 3). That was based on cDNAs encoding genes of interest synthesized using standard reverse transcription technology and printed on glass slides using an in-house automatic printer. RNA was reverse transcribed using labeled nucleotides and hybridized to cDNA. The intensity of fluorescence was read by a computerized array reader. This system was able to detect expression of 1024 genes in a sample. A commercial response was delivered early by Affymetrics, which developed a chip using 22–25 base oligonucleotides placed on a plastic surface and using 18–30 oligonucleotides per gene assayed. mRNA is reverse transcribed to obtain cDNA. The cDNA is then transcribed with biotin-labeled nucleotides to create biotin-labeled cRNA. Oligonucletide chips are hybridized with biotin-labeled cRNA and the abundance of specific transcript was obtained by reading the chip in a laser scanner. Total fluorescence obtained according to the complex algorithm from sense and missense probes is proportional to gene expression in the original sample. These two technologies (oligonucleotide array and cDNA microarray) have been developed in parallel and may be used in parallel or sequential fashion. Although both have advantages and drawbacks, they set up a standard in whole genome expression assessment. The following paragraph summarizes the basic aspects of both technologies and delivers a comparison of them. cDNA microarray (also known as a spotted array) was developed by groups led by Patrick Brown and Ronald Davis at Stanford University (Stanford, CA) (4, 5). The system is based on standard reverse transcription and PCR amplification followed by cloning of cDNA for each gene of interest. cDNA clones are then ‘printed’ on glass slides using a robotic printer. Several printing technologies were developed allowing, at least theoretically, to obtain an array with 100 000 spots. Each spot corresponds to one gene. Therefore, this system allows obtaining a customized array (6). mRNA obtained from biological samples using various methods is then reverse transcribed and the second strand of cDNA is also obtained in order to achieve double-stranded cDNA using primers end-labeled using fluorescent or radioactive tagged nucleotides and hybridized to the array. The fluorescence or radioactive emission from each ‘spot’ is proportional to the relative specific transcript abundance in the pool of cDNA hybridized to a cDNA spot. Abundance of a specific transcript in a sample is compared to the abundance of that transcript in a control sample. This technique, although laborious, delivers a high-density, completely customized system with high detection specificity. Materials to obtain a single set for spotted array are inexpensive, although robotic plasmid isolation systems, spot printer and scanner usually are parts of a core facility. The disadvantages comprise difficult clone handling and, in some cases, the system may not detect alternate splicing and does not correct for presence of SNPs in transcripts. Because the abundance of a transcript in a test sample is compared to that in a control sample, only relative abundance is determined. This technology was developed by Fodor in 1991 (7–9). It is patent protected by Affymetrics (Santa Clara, CA). In this system, oligonucleotide probes are directly synthesized on a solid surface using a chemical synthesis supported by a lithophotographic tech