Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons

Mao,Qiang; Zhang,Lujun; Guo,Yujie; Sun,Lu; Liu,Siwen; He,Peng; Huang,Rongzhong; Sun,Lin; Chen,Shigang; Zhang,Hong; Xie,Peng

doi:10.3892/mmr.2016.5959

Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons

Authors:
- Qiang Mao
- Lujun Zhang
- Yujie Guo
- Lu Sun
- Siwen Liu
- Peng He
- Rongzhong Huang
- Lin Sun
- Shigang Chen
- Hong Zhang
- Peng Xie
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Affiliations: Department of Neurology, Yongchuan Hospital, Chongqing Medical University, Chongqing 402460, P.R. China, Institute of Neuroscience and The Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016, P.R. China, Department of Rehabilitation, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, P.R. China, Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016, P.R. China
Published online on: November 22, 2016 https://doi.org/10.3892/mmr.2016.5959
Pages: 5587-5594

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Abstract

Borna disease virus (BDV) is a neurotropic RNA virus that infects the limbic system of mammals and results in behavioral disorders. The hippocampus is a core region in the limbic system, which contributes to memory and learning and is important in the regulation of emotion. However, no validated microRNA housekeeping genes have yet been identified in BDV‑infected rat primary hippocampal neurons. Proper normalization is key in accurate miRNA expression analysis. The present study used reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) to evaluate the expression stability of 10 commonly used reference genes [miR‑92a, 5S, U6, miR‑103, miR‑101a, miR-let-7a, miR‑16, E2 small nucleolar RNA (snoRNA), U87 and miR‑191] in BDV‑infected rat hippocampal neurons and non‑infected controls across 12 days post‑infection. The data was analyzed by four statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ‑Ct method. Subsequently, the most suitable reference genes (miR‑101a and U87) and the least suitable (snoRNA) were determined by the RankAggreg package. miR‑155 was selected as a standard by which to evaluate the most and least suitable reference genes. When normalized to the most stable reference gene there were significant differences between the two groups. However, when the data were normalized to the less stably expressed gene, the results were not significant. miR‑101a was recommended as a suitable reference gene for BDV-infected rat primary hippocampal neurons.

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