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Updated: Jul 9, 2026
Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis
Published on: May 16, 2020
Reference gene selection for real-time rtPCR in human epidermal keratinocytes
Danny Allen1, Eleanor Winters, Paul F Kenna
1Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. danny.allen@tcd.ie
Selecting appropriate housekeeping genes is crucial for accurate gene expression analysis using real-time reverse transcription quantitative PCR (RT-qPCR). This study found that the stability of these control genes varies significantly between human epidermal keratinocyte cell types, impacting RNA interference experiments.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- RNA interference (RNAi) is a key method for targeted mRNA suppression.
- Real-time reverse transcription quantitative PCR (RT-qPCR) is vital for measuring mRNA expression levels.
- Accurate RT-qPCR necessitates normalizing target gene expression to stable housekeeping genes.
Purpose of the Study:
- To determine the expression stability of 11 housekeeping genes in human epidermal keratinocytes.
- To assess how short hairpin RNA (shRNA) affects housekeeping gene expression profiles.
- To identify suitable control genes for RT-qPCR in different keratinocyte models.
Main Methods:
- Human epidermal keratinocytes (HaCaT, adult NHEK, juvenile NHEK) were utilized.
- Cells were transfected using lipid-based methods or AMAXA nucleofection.
- Real-time RT-qPCR and geNORM software were employed for expression analysis.
Main Results:
- For HaCaT and adult NHEK cells, 8 out of 11 housekeeping genes showed stable expression.
- For juvenile NHEK cells, only 4 housekeeping genes were sufficiently stable for normalization.
- Housekeeping gene expression can be influenced by RNA interference pathway induction.
Conclusions:
- Characterizing housekeeping gene stability in specific cell types is essential before RT-qPCR analysis.
- This research provides critical data for researchers using human epidermal keratinocytes.
- The methodology is broadly applicable to RT-qPCR studies across various cell types.
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