custom automated version of the aminoallyl MessageAmp II kit from Ambion.
Hybridization protocol
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.
Scan protocol
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.
Description
R. Kittler, V. Surendranath, A.-K. Heninger, M. Slabicki, M. Theis, G. Putz, K. Franke, A. Caldareli, H. Grabner, K. Kozak, J. Wagner, E. Rees, B. Korn, C. Sachse, B. Sonnichsen, J. Guo, J. Schelter, J. Burchard, P.S. Linsley, A.L. Jackson, B. Habermann, and F. Buchholz, Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies, 2007, Nature Methods
Data processing
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.
