Affymetrix GeneChip technology and bio-informatics

RNA was purified using the RNeasy Mini Kit (QIAGEN, Valencia, CA) followed by on-column DNA digestion with RNase-Free DNase (QIAGEN). RNA integrity was confirmed using the RNA 6000 Nano LabChip (Agilent Biotechnologies, Palo Alto, CA). Two replicate cRNA targets were generated from each HEC-1-A sub-line RNA preparation; all eight targets were made at the same time. Total cellular RNA (8 ug) was converted to cDNA using a T7-(deoxythymidine)₂₄ primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). Resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA; the latter was purified on an RNeasy spin column (QIAGEN) and chemically fragmented to a size range of 35 to 200 bp. cRNAs were concurrently hybridized to HG-U133A GeneChips (Affymetrix, Santa Clara, CA). Hybridizations were performed for 16 hours, followed by incubations with streptavidin-conjugated phycoerythrin, and polyclonal anti-streptavidin antibody coupled to phycoerythrin. GeneChips were scanned using an Agilent GeneArray laser scanner and images analyzed using Affymetrix MAS 5.0 software. Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA targets.

Unsupervised nearest-neighbor hierarchical clustering (Spotfire DecisionSite, Somerville, MA) identified a significant effect of culture condition/date of RNA collection on overall gene expression profiles. Therefore, to identify candidate KLF9 gene targets, the following was separately performed on the combinations of 4S/2AS and 9S/3AS. Intensity values of probe sets were imported into GeneSpring Gx 7.3 software for analysis. Values were processed using the Robust Multiarray Analysis algorithm for background adjustment, normalization and log₂-transformation of perfect match values. Data were subjected to per-chip and per-gene normalization and analyzed for differences between cell lines (fold-change, S relative to AS, value of 1.3 or higher; P < 0.05, Student's t test). Transcripts that passed these filters for both the 4S/2AS and 9S/3AS cell line combinations comprised the final KLF9-regulated gene lists and were compared for gene overlaps. The final gene list (comprised only of overlapping transcripts) was annotated using NETAFFX [24] and NCBI Entrez [25]. The microarray data have been deposited in Gene Expression Omnibus [26] as series GSE11855.