Abstract
Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.
Highlights
Borna disease virus (BDV) is the causative agent of Borna disease, an enzootic encephalomyelitis of horses and sheep named after epidemics having occurred in horses close to the city of Borna in Saxony (Germany) at the end of the 19th century
The standard curve revealed the amplification efficiencies for all candidate reference genes (Table 1). These results indicated that the method of measurement was appropriate
In order to validate the appropriate reference genes in these cells infected with a human BDV strain, we analyzed the expressions of 10 commonly-used candidate reference genes (HPRT, YWHAZ, TPB, Rpl13A, GAPDH, ACTB, PPIA, ARBP, 18sRNA and B2M)
Summary
Borna disease virus (BDV) is the causative agent of Borna disease, an enzootic encephalomyelitis of horses and sheep named after epidemics having occurred in horses close to the city of Borna in Saxony (Germany) at the end of the 19th century. BDV is a neurotropic, non-cytolytic, non-segmented, negative-stranded RNA virus belonging to the order, Mononegavirales. The BDV genome spans approximately 8.9 k band consists of six major open reading frames (ORFs). It is a neurotropic RNA virus that can infect many vertebrate species [1], including man. Whether or not BDV is involved in human disease, like mental disorders, remains a controversial issue [2]. BDV infection has been reported in a range of animal species across a broad global geographic distribution [3], including
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