Expression profiling by high throughput sequencing
Summary
The use of gene expression signatures to classify compounds, identify efficacy or toxicity, and differentiate close analogs relies on the sensitivity of the method to identify modulated genes. We used a novel ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq®, to determine whether previously unreported compound-responsive genes could be identified and incorporated into a broad but specific compound signature. TempO-Seq exhibits 99.6% specificity, single cell sensitivity, and excellent correlation with fold differences measured by RNA-Seq (R2 = 0.9) for 20,629 targets. Unlike many expression assays, TempO-Seq does not require RNA purification, cDNA synthesis, or capture of targeted RNA, and lacks a 3′ end bias. To investigate the sensitivity of the TempO-Seq assay to identify significantly modulated compound-responsive genes, we derived whole transcriptome profiles from MCF-7 cells treated with the histone deacetylase inhibitor Trichostatin A (TSA) and identified more than 9,000 differentially expressed genes. The TSA profile for MCF-7 cells overlapped those for HL-60 and PC-3 cells in the Connectivity Map (cMAP) database, suggesting a common TSA-specific expression profile independent of baseline gene expression. A 43-gene cell-independent TSA signature was extracted from cMAP and confirmed in TempO-Seq MCF-7 data. Additional genes that were not previously reported to be TSA responsive in the cMAP database were also identified. TSA treatment of 5 cell types revealed 1,136 differentially expressed genes in common, including 785 genes not previously reported to be TSA responsive. We conclude that TSA induces a specific expression signature that is consistent across widely different cell types, that this signature contains genes not previously associated with TSA responses, and that TempO-Seq provides the sensitive differential expression detection needed to define such compound-specific, cell-independent, changes in expression.
Overall design
Commercially available human reference and tissue RNAs, unpurified cell lysates from MCF-7 and MDA MB 231 cells, and synthetic ERCC RNAs were used to assess performance of a targeted whole transcriptome expression profiling method. In addition, MCF-7 cells, PC3 cells and HL60 cells were exposed to Trichostatin A, then lysed and profiled, to define a novel TSA-specific expression signature.
Samples GSM2429258 - GSM2429287 were purified reference RNAs and brain RNAs and mixtures between them that were run in the TempO-Seq assay in replicates of 6 each. Samples GSM2429258 - GSM2429287 have no associated raw data due to data loss.
The GSM2460368-GSM2460377 samples represent human reference RNA replicates that were processed in parallel, pooled in a single library and sequenced twice on the same instrument.
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