After amplification samples are put through a controlled fragmentation to improve hybridization sensitivity and consistency. The building of the fragmentation plate is completed using automated liquid handling and tracked by LIMS. Fragmented target is mixed with hybridization buffer prior to array hybridization.
GeneChip microarrays are loaded with the fragmented target sample/hybridization buffer mix using standard manual techniques. Arrays are hybridized for 18 hours at 45°C with vigorous mixing. Sample and array matching as well as hybridization times are recorded and monitored by the LIMS.
Labeling and Scanning
Sample labeling is completed after the amplified target is hybridized to the GeneChip. Unbound sample is removed and staining is accomplished through the binding of streptavidin conjugated phycoerythrin to the hybridized target. Excess label is removed and the array is ready for scanning. All of the washing and staining steps are performed by the Affymetrix FS450 fluidics station using standard protocols. Arrays are scanned using a GeneChip Scanner 3000 7G with a 48 array autoloader that allows the continuous adding of arrays during scan runs. This scanner also maintains the optimal temperature for the arrays prior to and during scanning.