Validation of Stable Housekeeping Genes for Quantitative Real Time PCR in Golden Syrian Hamster

Anim Models and Exp Med

Kang

Lan

et al. 2023

BackgroundAcute pancreatitis (AP) is a severe disorder that leads to high morbidity and mortality. Appropriate reference genes are important for gene analysis in AP. This study sought to study the expression stability of several reference genes in the golden Syrian hamster, a model of AP.MethodsAP was induced in golden Syrian hamster by intraperitoneal injection of ethanol (1.35 g/kg) and palmitoleic acid (2 mg/kg). The expression of candidate genes, including Actb, Gapdh, Eef2, Ywhaz, Rps18, Hprt1, Tubb, Rpl13a, Nono, and B2m, in hamster pancreas at different time points (1, 3, 6, 9, and 24 h) posttreatment was analyzed using quantitative polymerase chain reaction. The expression stability of these genes was calculated using BestKeeper, Comprehensive Delta CT, NormFinder, and geNorm algorithms and RefFinder software.ResultsOur results show that the expression of these reference genes fluctuated during AP, of which Ywhaz and Gapdh were the most stable genes, whereas Tubb, Eef2, and Actb were the least stable genes. Furthermore, these genes were used to normalize the expression of TNF‐α messenger ribonucleic acid in inflamed pancreas.ConclusionsIn conclusion, Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Stability Assessment Of Reference Genesmentioning

confidence: 99%

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Identification of optimal reference genes in golden Syrian hamster with ethanol‐ and palmitoleic acid‐induced acute pancreatitis using quantitative real‐time polymerase chain reaction

Anim Models and Exp Med

Kang

Lan

et al. 2023

“…Total RNA was extracted from tissues homogenates and purified with the TRIzol reagent according to the manufacturer's protocol. 7 Total RNA (1 μg) was reverse transcribed by PrimerScript RT Reagent Kit (TaKaRa, Japan) following manufacturer instructions. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) reactions were performed using the TB Green Premix Taq II (TaKaRa, Japan) following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

COVID-19 Resulting in Potential Hearing Damage of Rodents

Chinese medicine and natural products

Tian

et al. 2022

Objectives To find out the association between the sensorineural hearing loss and coronavirus disease 2019 (COVID-19), the expression of ACE2 and TMPRSS2 in hamsters and mice was detected. Design Using the public data from the National Center for Biotechnology Information and the Global Initiative on Sharing All Influenza Data, the expression of ACE2 and TMPRSS2 at the transcriptomic, DNA, and protein levels of ACE2 in the brain, inner ear, and muscle from the golden Syrian hamsters (Mesocricetus auratus) and mice (Mus musculus) was assessed. Results We identified ACE2 and TMPRSS2 expressed at different levels in the inner ear and brain at DNA and transcriptomic levels of both mice and hamsters. The protein expression from the brain and inner ear showed a similar pattern, while the expression of ACE2 from the inner ear was relatively higher than that from the muscle. Conclusion Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shows genetic potential to infect the hearing system of rodents and lead to sudden sensorineural hearing loss that can be used as a characteristic to detect asymptomatic patients of COVID-19.

“…Quantitative PCRs were carried out in triplicate using the specific primers for different immune cells that were previously described 16 and chemokine and cytokine (Table 2). The reference gene Syrian hamster RPL18 andβ‐actin gene were used for normalization as previously described 16,21 . All reactions were performed twice to ensure the technical reproducibility of the assays.…”

Section: Methodsmentioning

confidence: 99%

Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system

Anim Models and Exp Med

Lan

Guo

et al. 2022

BackgroundSHARPIN (SHANK‐associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF‐κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system.MethodsA single‐guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase‐quantitative polymerase chain reaction.ResultsAll the offspring harbored germline‐transmitted SHARPIN mutations. Compared with wild‐type hamsters, SHARPIN protein was undetectable in SHARPIN−/− hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN−/− hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF‐κB and phosphorylation of NF‐κB and IκB protein significantly diminished in SHARPIN−/− animals.ConclusionsA novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.