A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling

Abstract

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.

Extraction-Free Whole Transcriptome Gene Expression Analysis of FFPE Sections and Histology-Directed Subareas of Tissue

“…The TempO-Seq assay for FFPE samples relies on the standard TempO-Seq chemistry (7)(8)(9)(10)(11). The assay ( Figure 1) was modified for FFPE samples as follows: An area of interest on a slidemounted FFPE section ( Figure 1A) is scraped from the slide and deposited directly into BioSpyder 1X FFPE lysis buffer ( Figure 1B).…”

“…The assays used were the human whole transcriptome assay (7) which measures 19,283 genes (21,111 probes); the mouse whole transcriptome assay which measures 23,580 genes (30,147 probes); and the rat whole transcriptome assay which measures 21,119 genes (22,253 probes). Each gene is measured by one or more probes formed by ligation of a DO pair, as previously described (7).…”

Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis via SYK activation

“…due to clonal expansion), we performed a targeted transcriptomics analysis using the TempO-Seq technology ( Figure 5A). 50 Only seven out of 21112 of the identified transcripts (0.03%) were significantly altered in FES S700C compared to WT HL-60 macrophages, which indicates that introduction of this mutation minimally disturbs gene expression.…”

The basal level of gene expression associated with chromatin loosening shapes Waddington landscapes and controls cell differentiation

“…A signature analysis was then conducted to determine the transcriptomic changes induced by TSA in MCF7 cells. analysed from the data of [45]. (D) Parallel effects of DP-MRTFA expression (DP) and TSA treatment on relative changes obtained in transcriptomic arrays for selected gene markers (maximal signals set to 1).…”

“…MCF7 cells. Data were obtained from by TempO-Seq targeted whole transcriptome profiling GEO accession: GSE91395 [45].…”