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

Abstract

Background and aim of studyqPCR is a robust technique which quantifies the expressions of target genes in relation to reference genes. Stresses such as virus infection or heat shock change expressions of many cellular genes including the reference genes, so the aim was to introduce a constant calibrator to normalize the data to.MethodologyConstructed glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plasmid was transcribed to GAPDH RNA and used as spike RNA. Spiked RNA samples were subjected to qPCR at different conditions such as virus infection, IFN treatment, or mild heat shock.The resultsAdenovirus 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.ConclusionThe internal controls that were chosen for this study completely changed the experimental results since they were affected with the experimental conditions. However, GAPDH spike RNA level was stable in its amplification at different kinds of stresses. So it can be an alternative for housekeeping gene due to its stability at these different conditions.