Microarray Comperative genomic hybridization - Human MDA-MB-453

DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.

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Protocol tips
Slight modifications were introduced WGA products processed with the PCR-based labeling approaches. Here, the hybridization mix consisted of 5.0 µg of Cot1-DNA (Roche Diagnostics), 12 µl of 10x Blocking Reagent (Agilent Technologies), 60 µl of 2x Hi RPM Hybridization Buffer, 1% (v/v) of both Tween20 and Igepal and 19 µl of both test and reference DNA. For each hybridization 100 µl of the hybridization mix was applied on the array and hybridized at 65°C for 24 h. Following the hybridization, the slides were washed twice for 2:30 min in Oligo aCGH/ChIP-on-Chip Wash Buffer 1 (Agilent Technologies) at room temperature, twice for 30 sec min in Oligo aCGH/ChIP-on-Chip Wash Buffer 2 (Agilent Technologies) at 37°C. Washed slides were immersed in acetonitrile to remove all remaining traces of the wash buffers. Finally, slides were scanned using an Agilent Microarray Scanner Type C.
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