DNA Microarray Technology

Comprehensive reviews of microarray techniques are available.1-3 DNA microarrays are orderly, high-density arrangements of nucleic acid gene segments (either spotted complementary DNAs [cDNAs] or oligonucleotides), which enable broad-scale gene expression profiling. Labeled probes are generated from RNA by either reverse transcription or an in vitro transcription reaction using cDNA as a template and incorporating a specific label (Figure 29-1). The labeled probes are hybridized to the gene array, and relative signals detected, quantified, and compared.

cDNA Arrays cDNA arrays are produced by spotting individual cDNA probes (usually purified PCR products) onto a solid support (nylon membrane or glass slide) using a robotic device, such that the entire array of spots represents a cDNA library. The cDNA array is used to compare the gene expression patterns of two conditions, such as normal compared to diseased tissue, benign compared to malignant tissue, or treated compared to untreated states. The RNA populations from the two comparative conditions are labeled with two different fluorescently labeled Cy-3 or Cy-5 tagged nucleotides. The two RNA-derived labeled probe sets are hybridized to separate arrays, or pooled in equal

hybridized to the microarray, which then is scanned. The expression values are derived from the resulting hybridized probe signals, providing raw data for analysis.

amounts and hybridized to the same array. The fluorescent signals are measured and displayed as a ratio of the two fluorescent signals.

Oligonucleotide Arrays

Oligonucleotide arrays use small DNA sequences (20 to 70 nucleotides in length), either synthesized chemically and spotted onto a solid support or synthesized directly on the solid support or chip, such that the entire array of spots represents a set of genes. The most common version of this type of microarray is produced commercially by Affymetrix (Santa Clara, CA). In this version, the short oligonucleotides (length varies depending on the version of the chip) are synthesized in situ on the chip, with each gene represented by several oligonucleotides distributed along the length of the gene plus separate oligonucleotides with a single base mismatch with the gene sequence and used as a negative background control. Unlike spotted cDNA arrays, the two RNA populations to be compared are fluorescently labeled and hybridized to separate chips, and the fluorescent signal is detected. Control complementary RNAs (cRNAs) are spiked into each RNA population probe and used as an internal hybridization control. The supplied software converts the fluorescent intensity into a signal value for each gene, which is directly related to the amount of target mRNA in the probe. The relative signals for the same gene in the two RNA populations are then compared.

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