TPD-seq: A high throughput RNA-seq method to derive transcriptomic points of departure from cell lines

Area of Science:

• Toxicology
• Genomics
• Computational Biology

Background:

• Growing interest in transcriptomic points of departure (tPOD) from high-throughput in vitro experiments.
• Need for democratized tPOD research methods.
• Existing methods require advanced bioinformatics expertise.

Purpose of the Study:

• To outline and validate the TPD-seq workflow for deriving tPOD values.
• To link microplate-based cell exposures with RNA-seq and bioinformatics analysis.
• To demonstrate TPD-seq utility with common toxicity testing chemicals.

Main Methods:

• TPD-seq workflow: microplate cell culture (Caco-2, Hep G2, RTgill-W1), commercial RNA-seq kit, cloud-based bioinformatics (ExpressAnalyst.ca).
• Application to solvents (DMSO, methanol) and positive controls (DCA, H2O2).
• Analysis of gene expression data, curve-fitting, and gene benchmark dose calculation.

Main Results:

• Successful mapping of RNA-seq reads to protein-coding genes.
• Significant percentage of differentially expressed genes yielded gene benchmark doses.
• Derived tPOD values for DMSO (31-155 mM), DCA (~5-76 μM), and H2O2 (0.7-1.1 mM in Caco-2, 5-30 μM in Hep G2).
• DMSO exposure yielded the most robust transcriptomic responses.

Conclusions:

• TPD-seq provides a scalable and accessible workflow for tPOD derivation.
• The method is suitable for laboratories with basic molecular and cell culture facilities.
• Further case studies can determine the full performance and scalability of TPD-seq.