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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

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A comparison of the TempO-Seq and Affymetrix microarray platform using RTqPCR validation.

Matthias M Wehr¹, Stella Marie Reamon-Buettner², Detlef Ritter²

¹Fraunhofer Institute for Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany. Matthias.wehr@item.fraunhofer.de.

|July 3, 2024

Summary

Comparing TempO-Seq and Affymetrix platforms for transcriptome analysis revealed significant differences in differentially expressed genes (DEGs). However, both methods showed high concordance in gene regulation and were validated by RTqPCR, supporting their use in toxicological risk assessment.

Keywords:

Affymetrix Chemical perturbation Microarray RT2 profiling arrays RTqPCR TempOSeq Transcriptomics

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Last Updated: Jun 22, 2025

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Area of Science:

Toxicology and Molecular Biology
Next-generation risk assessment utilizing mechanistic data
Application of new approach methods (NAMs) in toxicology

Background:

Transcriptome data is crucial for next-generation risk assessment using new approach methods.
High-throughput sequencing and microarray technologies identify differentially expressed genes (DEGs).
Integrating data from different technologies requires understanding platform-specific differences.

Purpose of the Study:

To compare the TempO-Seq platform and Affymetrix chip technology for whole-genome transcriptome analysis.
To evaluate the concordance and differences in identifying differentially expressed genes (DEGs).
To assess the suitability of RTqPCR for validating results from both platforms in toxicological studies.

Main Methods:

Whole-genome transcriptome data analysis comparing TempO-Seq and Affymetrix platforms.
Exposure of cells to the volatile compound dimethylamine.
Selection and validation of differentially expressed genes (DEGs) using RTqPCR.

Main Results:

The overlap of differentially expressed genes (DEGs) between TempO-Seq and Affymetrix platforms was less than 37%.
A high concordance was observed in the log2fold changes (gene regulation) between the two platforms.
RTqPCR validated the majority of DEGs from both platforms, with minor conflicts and non-detections.

Conclusions:

Despite platform-specific differences in DEG identification, both TempO-Seq and Affymetrix are suitable for toxicological applications.
RTqPCR serves as a reliable validation method for transcriptome data generated by these platforms.
Understanding platform variations is essential for integrating multi-platform transcriptome data in risk assessment.