By Hanm Brezis
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In order to obtain good results, the spots on the array should contain uniform, equivalent amounts of spotted polymerase chain reaction (PCR) products or oligonucleotides. Pre-scanning the slide before using it in a hybridization experiment can verify this property. , 2001]. Alternatively, Shearstone and colleagues have described a method for printing oligonucleotide microarrays spiked with small amounts of dCTPCy3 or dCTP-Cy5. , 2002]. 3 Quality control during array manufacturing Many institutions have established core facilities that manufacture microarrays by spotting either PCR products from cDNA clones or chemically synthesized oligonucleotides on glass slides.
In this section, we describe how to monitor the quality of two critical ingredients—the RNA samples and the microarray slides—before the hybridization step. Because assessment of hybridization quality is typically based on the scanned image, we will reserve its discussion to a later section. 1 Monitoring RNA quality The basic component of a microarray experiment is the RNA extracted from samples. Before running a microarray experiment, this quality should be tested. If enough total RNA is available, it can be evaluated by agarose gel electrophoresis with ethidium bromide.
4 Signal-to-noise ratio One of the most effective measures for the overall quality of a microarray experiment is the percentage of spots with adequate signal-to-noise ratio (S/N). The precise definition of “adequate” in this setting may vary from platform to platform. 0 provides detection p- 34 Coombes et al. values along with “present” or “absent” calls for each gene. We typically find that around 40–50% of the genes are present in a successful hybridization on an Affymetrix microarray. On our own two-color fluorescent microarrays, we typically require good spots to have S/N > 2.