Evaluating the applicability of mouse SINEs as an alternative normalization approach for RT-qPCR in brain tissue of the APP23 model for Alzheimer’s disease

Janssens, Jonathan; Crans, René A. J.; Craenenbroeck, Kathleen Van; Vandesompele, Jo; Stove, Christophe; Dam, Debby Van; Deyn, Peter Paul De

doi:10.1016/j.jneumeth.2019.03.005

Cited by 8 publications

(8 citation statements)

References 75 publications

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“…Two microliters of cDNA was subjected in duplicate to quantitative PCR (qPCR) using LightCycler® 480 SYBR Green I Master (#04707516001, Roche) and specific primers for ACE2 42 and TMPRSS2 (Table S5). Beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and YWHA were included as housekeeping genes for the normalization of gene expression 43 . No template sample was used as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Organ-specific genome diversity of replication-competent SARS-CoV-2

et al. 2021

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease.

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Section: Methodsmentioning

confidence: 99%

Organ-specific genome diversity of replication-competent SARS-CoV-2

et al. 2021

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“…Analysis of GLT-1 mRNA expression was performed in cortical tissue derived from animals that were not included into the susceptibility study. All analyses were performed on left hemispheric cortices (n = 30) from HET and WT animals aged 3, 6 and 12 months, according to Janssens et al (Janssens et al, 2019). In short, RNA extraction was performed using the RNeasy® Plus Universal Mini kit (Qiagen, Hilden, Germany) and on-column DNase treatment was performed using the RNase-free DNase set (Qiagen, Hilden, Germany) following the manufacturer's instructions.…”

Section: Reverse Transcription Quantitative Polymerase Chain Reaction (Rt-qpcr)mentioning

confidence: 99%

Pentylenetetrazole-induced Seizure Susceptibility in the Tau58/4 Transgenic Mouse Model of Tauopathy

et al. 2020

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“…Different algorithms provide slightly different results [2,14,15]. To escape that bias, this paper uses a novel approach by calculating a comprehensive reference gene stability ranking based on four algorithms [3,7,[15][16][17][18][19]. Among different reference genes, Gapdh, Actb, Ppia, B2m, Hprt1, Ywhaz, Rpl13a, Pgk1, and Sdha are widely used in RT-qPCR experiments on brain tissue or brain-derived cell lines in laboratory rats.…”

Section: Ptz Model Of Seizuresmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures

et al. 2020

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Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20–22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

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Evaluating the applicability of mouse SINEs as an alternative normalization approach for RT-qPCR in brain tissue of the APP23 model for Alzheimer’s disease

Cited by 8 publications

References 75 publications

Organ-specific genome diversity of replication-competent SARS-CoV-2

Organ-specific genome diversity of replication-competent SARS-CoV-2

Pentylenetetrazole-induced Seizure Susceptibility in the Tau58/4 Transgenic Mouse Model of Tauopathy

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures

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