custom automated version of the aminoallyl MessageAmp II kit from Ambion.
Microarrays are incubated at 40°C for 48 hours in a rotating carousel. Hybridizations to custom Agilent microarrays are completed as previously described (Hughes et al.Nat Biotech (2001), 19(4):342-7). Microarrays are washed to remove non-specific hybridized sample. Afterwards, microarrays are dried in an ozone-free nitrogen chamber.
Microarrays are scanned using the Agilent LP2 laser scanner. The scanner output is a Tiff file, which contains the quantitative hybridization data from each individual microarray. The Tiff files are then processed using Rosetta custom feature extraction software.
Linsley PS, Schelter J, Burchard J, Kibukawa M, Martin MM, Bartz SR, Johnson JM, Cummins JM, Raymond CK, Dai H, Chau N, Cleary M, Jackson AL, Carleton M, and Lim L. MCB (2007).
Data were processed using the Rosetta Resolver® system. Rosetta's custom feature extraction software performs error modeling before data are loaded into the Resolver system. The Resolver system performs a squeeze operation that combines replicates of the same sequence in an array while applying error weighting. The error weighting consists of adjusting for additive and multiplicative noise. A P-value is generated and propagated throughout the system. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds, below which genes of a P-value are considered to be significantly expressed. The Resolver system also combines multiple arrays using a squeezing process. If multiple spots reference one sequence, summarization is performed using an error-weighted average as described in Roland Stoughton and Hongyue Dai, Statistical Combining of Cell Expression Profiles. US Patent #6,351,712, February 26, 2002.