Gene · 2025
Reference genes for quantitative real-time polymerase chain reaction in in vitro non-alcoholic fatty liver disease.
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Chen Jingzhi, Chen Xingmi, Liu Ying, Lyu Chunming, Xu Ming, Yang Yang
Аннотация
BACKGROUND: In metabolic dysfunction-associated fatty liver disease (MAFLD) research, reference genes for qPCR are crucial but often unvalidated.
METHODS: RNA-seq was performed in control and free fatty acid (FFA) treated AML12 cells, and the candidates for reference gene were selected by previous literatures and filtered by dual-index ranking via normalization of coefficient variation and Log2FoldChange in the RNA-seq library. qPCR data was further analyzed via NormFinder, BestKeeper, geNorm, ΔCt and RefFinder to assess reference gene expression stability.
RESULTS: FFA treated cells showed a significantly increased lipid droplet accumulation. RNA-seq dual-index ranking and RefFinder identified that Ywhaz was the most stable reference gene.
CONCLUSION: Our finding revealed that Ywhaz is the most stable reference gene for qPCR in the AML12 cell model of FFA-induced MAFLD, and provided a reliable procedure for screening reference genes.
METHODS: RNA-seq was performed in control and free fatty acid (FFA) treated AML12 cells, and the candidates for reference gene were selected by previous literatures and filtered by dual-index ranking via normalization of coefficient variation and Log2FoldChange in the RNA-seq library. qPCR data was further analyzed via NormFinder, BestKeeper, geNorm, ΔCt and RefFinder to assess reference gene expression stability.
RESULTS: FFA treated cells showed a significantly increased lipid droplet accumulation. RNA-seq dual-index ranking and RefFinder identified that Ywhaz was the most stable reference gene.
CONCLUSION: Our finding revealed that Ywhaz is the most stable reference gene for qPCR in the AML12 cell model of FFA-induced MAFLD, and provided a reliable procedure for screening reference genes.