GAPDH spike RNA as an alternative for housekeeping genes in relative gene expression assay using real-time PCR

Abstract

Adenovirus hexon in interferon-deficient cells showed different expression levels when data were normalized to GAPDH or 18S. Consistently, hexon expression levels were different in untreated cells under the control or heat-shocked conditions when data were normalized to GAPDH or 18S. Promyelocytic leukemia protein II (PML-II) expression level was lower in HeLa-PML-II-deficient cells (PML-II-Kd) compared to the control when the data were normalized to GAPDH as a reference gene and also in GAPDH RNA spiked, which showed reasonable consistency. More consistent data were obtained when the GAPDH normalizer was added before the step of treating the extracted RNA with DNase compared to add it after the treatment or directly to the qPCR reaction.