Reference Genes for Expression Studies in Human CD8⁺ Naïve and Effector Memory T Cells under Resting and Activating...

Area of Science:

• Immunology
• Molecular Biology
• Gene Expression Analysis

Background:

• Accurate mRNA quantification using reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) relies on stable reference genes.
• While reference genes are validated for bulk T cells, specific subsets like naïve (NV) and effector memory (EM) CD8+ T cells lack such validation, despite differential responses to activation.

Purpose of the Study:

• To evaluate the stability of twelve commonly used reference genes in human NV and EM CD8+ T cells under non-activated and activated conditions.
• To identify reliable reference genes for accurate gene expression analysis in these specific T cell subsets.

Main Methods:

• Human naïve and effector memory CD8+ T cells were subjected to activation for 2, 10, and 20 hours.
• Twelve candidate reference genes were analyzed for expression stability using five statistical methods.
• Expression levels of candidate reference genes were quantified using RT-qPCR.

Main Results:

• Several commonly used reference transcripts showed differential expression upon T cell activation.
• The stability of reference genes significantly impacted the quantification of other transcripts, as demonstrated with IFNG mRNA.
• 18S rRNA and SDHA mRNA were identified as the only stable reference gene candidates across all tested conditions.

Conclusions:

• The use of inappropriate reference genes can lead to inaccurate mRNA quantification in activated T cell subsets.
• 18S rRNA and SDHA mRNA are recommended as stable reference genes for RT-qPCR studies involving human naïve and effector memory CD8+ T cells under activation.
• This finding is critical for reliable gene expression profiling in T cell immunology research.