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Beyond Affymetrix arrays: expanding the set of known hybridization isotherms and observing pre-wash signal intensities. | LitMetric

AI Article Synopsis

  • - The study investigates how probe saturation and post-hybridization washing affect the accuracy of target abundance measurements in microarray hybridization isotherms.
  • - Researchers examined hybridization isotherms using ribosomal RNA on various microarray platforms, discovering different shapes and behaviors in the signals generated.
  • - Results indicated that fluorescent signals from probes hybridized to the same target varied significantly, influenced by factors like hybridization time and probe sequence, highlighting the complexity of hybridization and washing processes.

Article Abstract

Microarray hybridization studies have attributed the nonlinearity of hybridization isotherms to probe saturation and post-hybridization washing. Both processes are thought to distort 'true' target abundance because immobilized probes are saturated with excess target and stringent washing removes loosely bound targets. Yet the paucity of studies aimed at understanding hybridization and dissociation makes it difficult to align physicochemical theory to microarray results. To fill the void, we first examined hybridization isotherms generated on different microarray platforms using a ribosomal RNA target and then investigated hybridization signals at equilibrium and after stringent wash. Hybridization signal at equilibrium was achieved by treating the microarray with isopropanol, which prevents nucleic acids from dissolving into solution. Our results suggest that (i) the shape of hybridization isotherms varied by microarray platform with some being hyperbolic or linear, and others following a power-law; (ii) at equilibrium, fluorescent signal of different probes hybridized to the same target were not similar even with excess of target and (iii) the amount of target removed by stringent washing depended upon the hybridization time, the probe sequence and the presence/absence of nonspecific targets. Possible physicochemical interpretations of the results and future studies are discussed.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836560PMC
http://dx.doi.org/10.1093/nar/gkp1122DOI Listing

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