Rice Genetics IV - IRRI books - International Rice Research Institute

duction of chips is done commercially, requiring specialized equipment and proceduresand chip array-specific software. Chips, at least at present, are not within themeans of most academic research laboratories, but the generation and use of DNAmicroarrays are.The production of DNA microarrays includes the selection and preparation of theprobes and their robotic deposition on suitable surfaces (see Deyholos and Galbraith2001, for an in-depth discussion of the technology). Pursuing this technology requiresa long-term commitment in resources and training. Table 4 presents an over-Table 4. Microarray use and problems.Equipment and procedures aThe following are minimum equipmentnecessities, associated withconsiderable costPCR machinesSpotting robotMicroarray reader (scanner)Amplification of ESTs or cDNAsDNA purification and quantificationSpottingControl genesQuality controlHybridizationAnalysis of fluorescence imagesStatistical analysis and clusteringRemarksGeneral equipment category, which is essential.Best used when funds and equipment are sharedthrough a center. Permanent supervision required.Commercial kits; robots optional; PCR productsmay be amplified from bacterial colonies or fromplasmids; production and quality control are timeconsuming.Assembly of plates (96/384) is asource for error. Bar-coding and random checks byresequencing are recommended.Commercial kits; robots optional but required forlarge-scale work. Rearrangement of PCR productsmay be necessary to eliminate inefficientlyamplified products or those producing multiplebands.Training required; various treatments of slide surfacesfor DNA attachment; controlled environmentalconditions are essential during spotting/printing.Inclusion of positive and negative control genes.Negative controls are best taken from widelydivergent species (e.g., mammalian genes).Slides must be visually checked for irregularities.Background control; hybridization chambers must betested; ion precipitates must be avoided.Aberrant spots (off-center, dust particles, poor DNAretention) must be flagged and eliminated; timeconsumingstep.Require repeat hybridizations and multiple timepoints.aPCR = polymerase chain reaction, EST = expressed sequence tag.354 Bohnert et al