Two-color human Agilent 4×44K gene-expression arrays were used, as described by the manufacturer, comparing signal from control cells (Cy3-labelled) and test cells (Cy5-labelled). Array elements were filtered for those meeting confidence thresholds for spot size, architecture, and level above local background. These criteria are a feature of the Agilent gene expression software package for Agilent 4×44k arrays.
Agilent 4 44k gene expression arrays
Manufactured by Agilent Technologies
The Agilent 4 × 44K gene expression arrays are high-density microarray products designed for comprehensive whole-genome expression analysis. Each array contains 44,000 probes that target well-characterized genes and transcripts, providing a thorough coverage of the human, mouse, or rat transcriptome.
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5 protocols using agilent 4 44k gene expression arrays
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Two-color Agilent 4x44K gene expression
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Gene Expression Profiling of TLR9 and MyD88 Knockdown
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Cells were transduced with shRNAs, puro-selected and harvested at indicated times after shRNA induction. RNA was isolated using RNEasy mini kits (Qiagen). Gene expression was assessed using two-color human Agilent 4 × 44K gene expression arrays following the manufacturers protocol. Briefly, control shSC4 (control, Cy3-labelled) RNA was compared to RNA from cells with shRNAs targeting TLR9 (C4), TLR9 (D7), MyD88 (A7), MyD88 (B3) (Cy5-labelled) at each of the indicated time points. Array elements were filtered for spot quality using Agilent Feature Extraction software version 10.7, specific genes were determined to be downregulated if the log2 fold change (comparing control shSC4 to shRNA for TLR9) was less than −0.3 for at least 3 of the 4 time points (shTLR9) per cell line. Gene expression data have been deposited in Gene Expression Omnibus (GEO) under accession GSE99276. Signature enrichment was performed as previously described 32 (link). Briefly, downregulated genes were tested for overlap with published gene signatures in a 2×2 contingency table using a Fisher’s exact test.
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CTC Isolation and Transcriptome Analysis
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CTC isolation was performed from 7.5 ml of peripheral blood from advanced NSCLC patients or healthy donors using the EpCAM-based CELLectionTM Epithelial Enrich Dynabeads® kit (Invitrogen, Dynal; Oslo, Norway) according to manufacturers’ instructions. Isolated CTCs were resuspended in RNA later® (Ambion; Foster City, CA, USA) and preserved at −80°C until use.
Total RNA extraction, Complete Whole Transcriptome Amplification (WTA2, Sigma Aldrich) and gene expression array was performed as described by Barbazan et al.11 (link). Briefly, total RNA was extracted with the QIAmp viral RNA mini kit (Qiagen, Valencia, CA, USA) specifically designed for very low cellularity samples. Subsequent pure RNA was then subjected to Complete Whole Transcriptome Amplification PCR for 20 cycles using the maximum amount of RNA; Cy3 labelling and hybridization onto Agilent 4 × 44 k gene expression arrays. Upon hybridization, signal was captured and submitted to Significance Analysis for Microarray to identify differentially expressed genes (Supplementary Material and Methods S1 ).
Total RNA extraction, Complete Whole Transcriptome Amplification (WTA2, Sigma Aldrich) and gene expression array was performed as described by Barbazan et al.11 (link). Briefly, total RNA was extracted with the QIAmp viral RNA mini kit (Qiagen, Valencia, CA, USA) specifically designed for very low cellularity samples. Subsequent pure RNA was then subjected to Complete Whole Transcriptome Amplification PCR for 20 cycles using the maximum amount of RNA; Cy3 labelling and hybridization onto Agilent 4 × 44 k gene expression arrays. Upon hybridization, signal was captured and submitted to Significance Analysis for Microarray to identify differentially expressed genes (
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Gene Expression Profiling of TLR9 and MyD88 Knockdown
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Gene Expression Analysis of DLBCL Cell Lines Treated with ND-2158
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Four ABC DLBCL cell lines (OCI-Ly10, TMD8, HBL1, and OCI-Ly3), were treated with either 10 µM ND-2158 or 10 µM of the structurally related negative control compound ND-1659 for 6, 12, 24, or 36 h in culture. Cells were harvested at each time point, and RNA for gene expression was prepared from 5 million cells using TRIzol (Invitrogen) extraction, followed by clean up using the RNeasy kit (QIAGEN). Gene expression profiling was performed using two-color human Agilent 4 × 44K gene expression arrays (Agilent Technologies), as described by the manufacturer, comparing signal from control compound-treated (ND-1659) control cells (Cy3) with cells treated with ND-2158 (Cy5) for the indicated times. Array elements were filtered for those meeting confidence thresholds for spot size, architecture, and level above local background. These criteria are a feature of the Agilent Technologies gene expression software package for Agilent 4 × 44K arrays. Gene expression signature enrichment was performed by comparing changes in gene expression from at least two ABC DLBCL cell lines, occurring in at least two time points after ND-2158 treatment. Gene expression signature enrichment analysis was performed as described previously (Yang et al., 2012 (link)). Gene expression data has been deposited under GEO accession no. GSE63029 .
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