total RNA was prepared using TRIzol reagent as instructed and 10µg of total RNA or RNA extracted from an IP using 10µg of antibody was used per reaction
Label
Cy5
Label protocol
Total RNA quality was ascertained using an Agilent 2100 bioanalyzer (Agilent technologies). RNA from each sample and the reference (Universal Human Reference RNA, Stratagene) were hybridized to oligo (dT) primers at 65º C and then incubated at 42º C for 2 hours in the presence of reverse transcriptase, Cy5- or Cy3-dUTP and Cy5- or Cy3-dCTP, and a deoxynucleotide mix. In all cases, HeLa derived RNA samples were labeled with Cy5 and reference samples were labeled with Cy3.
Total RNA quality was ascertained using an Agilent 2100 bioanalyzer (Agilent technologies). RNA from each sample and the reference (Universal Human Reference RNA, Stratagene) were hybridized to oligo (dT) primers at 65º C and then incubated at 42º C for 2 hours in the presence of reverse transcriptase, Cy5- or Cy3-dUTP and Cy5- or Cy3-dCTP, and a deoxynucleotide mix. In all cases, HeLa derived RNA samples were labeled with Cy5 and reference samples were labeled with Cy3.
Hybridization protocol
In all cases, HeLa derived RNA samples were labeled with Cy5 and reference samples were labeled with Cy3. NaOH was used to destroy residual RNA. Sample and reference cDNA were then pooled, purified with QIAquick Purification Columns (Qiagen), mixed with hybridization buffer (50% formamide, 5 SSC, and 0.1% SDS), COT-1 DNA, and poly-deoxyadenylic acid to limit nonspecific binding, and heated to 95º C for 2 minutes. This mixture was pipetted onto a microarray slide, and hybridized overnight at 42º C on the MAUI hybridization system (BioMicro Systems). The array was then washed at increasing stringencies and scanned on a GenePix 4000B microarray scanner (Axon Instruments). All protocols are available in greater detail on the Duke Microarray Facility Web site (http://microarray.genome.duke.edu/services/spotted-arrays/protocols).
Arrays were printed at the Duke Microarray Facility using the Genomics Solutions OmniGrid 300 Arrayer. The arrays contain the Human Operon v3.0.2 arrays (Oligo Source) that possess 34,602 unique optimized 70-mers. RNA quality was ascertained using an Agilent 2100 bioanalyzer (Agilent technologies). RNA from each sample and the reference (Universal Human Reference RNA, Stratagene) were hybridized to oligo (dT) primers at 65º C and then incubated at 42º C for 2 hours in the presence of reverse transcriptase, Cy5- or Cy3-dUTP and Cy5- or Cy3-dCTP, and a deoxynucleotide mix. In all cases, HeLa derived RNA samples were labeled with Cy5 and reference samples were labeled with Cy3. NaOH was used to destroy residual RNA. Sample and reference cDNA were then pooled, purified with QIAquick Purification Columns (Qiagen), mixed with hybridization buffer (50% formamide, 5 SSC, and 0.1% SDS), COT-1 DNA, and poly-deoxyadenylic acid to limit nonspecific binding, and heated to 95º C for 2 minutes. This mixture was pipetted onto a microarray slide, and hybridized overnight at 42º C on the MAUI hybridization system (BioMicro Systems). The array was then washed at increasing stringencies and scanned on a GenePix 4000B microarray scanner (Axon Instruments). All protocols are available in greater detail on the Duke Microarray Facility Web site (http://microarray.genome.duke.edu/services/spotted-arrays/protocols).
Data processing
All arrays were subject to background subtraction followed by loess normalization within each array and scale normalization across all arrays using the arrayMagic package in R (Buness A., Huber W., Steiner K., Sueltmann H., Poustka A. arrayMagic: two-colour cDNA microarray quality control and preprocessing. Bioinformatics 2005 21, 554–556). KNN impute package in GenePattern (Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP (2006) GenePattern 2.0 Nature Genetics 38 no. 5 (2006): pp500-501 doi:10.1038/ng0506-500) was used to impute missing data if a probe had intensity values for at least half the samples. Otherwise the probes were excluded from analysis. Replicate probes were collapsed to one probe corresponding to the median value of all the replicates.
