Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies. This manuscript describes the adaptation of an alternative RT-qPCR-based 3′:5′ assay as an additional option for the quantitative assessment of rat RNA integrity. Two PCR primer sets were designed on the 3′ and 5′ regions of a rat housekeeping gene to evaluate RNA integrity by measuring the relative expression (3′:5′ ratio) of these amplicons. The 3′:5′ ratios were then compared to Agilent Bioanalyzer’s RNA integrity number (RIN) for a wide range of RNA samples originating from different tissues, cultured cell lines and rat strains that were prepared freshly, stored for years at −80 °C, purchased commercially or intentionally degraded. The 3′:5′ ratios and RIN values presented similar assessment of RNA integrity status from intact to heavily degraded samples. Based on the LOWESS regression of this large comparison dataset, 3′:5′ ratio threshold criteria equivalent to RIN cut-off values can be proposed for the selection of RNA samples for RT-qPCR analyses. This qPCR-based assay is easy to implement, cost-effective, and provides a reliable quantification of RNA integrity to assist in the selection of rat RNA samples suitable for downstream RT-qPCR gene expression analyses.
Background:Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by dysfunction of exocrine glands secondary to lymphocytic infiltration. Lymphoid tyrosine phosphatase (LYP) regulates T and B lymphocyte activation.PTPN22gene encodes LYP; multiple polymorphic variants have been described as genetic risk factor of autoimmune diseases.Objectives:The aim was to analyze thePTPN22rs2488457G>C, rs33996649G>A, and rs2476601C>T genetic variants relationship with the development risk of pSS in the western Mexico population.Methods:One hundred and eighty healthy subjects (HS) and 150 pSS patients, classified according to EULAR 2016 criteria, were included. The genetic variants and mRNA expression were determined through PCR-RFLP and qPCR assays.Results:The frequency of heterozygote rs33996649GA genotype was higher in pSS patients than HS [OR=3.143 (1–10.234), p=0.046], and also, rs33996649GA genotype was associated with high SSDAI score (p=0.01). The pSS patients showed 44-fold more mRNA expression in comparison with HS (p=0.002), and mRNA expression correlates with SSDAI (r2=0.512, p=0.006).Conclusion:The rs33996649G>A genetic variant of thePTPN22gene is associated with increased development risk of pSS in the western Mexican population. The expression mRNA correlates with disease activity in pSS.References:[1]Brito-Zerón, P., Baldini, C., Bootsma, H., Bowman, S. J., Jonsson, R., Mariette, X., Ramos-Casals, M. (2016). Sjögren syndrome.Nature Reviews Disease Primers, 2(July), 1–20.https://doi.org/10.1038/nrdp.2016.47[2]Stanford, S. M., & Bottini, N. (2014). PTPN22: The archetypal non-HLA autoimmunity gene.Nature Reviews Rheumatology,10(10), 602–611.https://doi.org/10.1038/nrrheum.2014.109[3]Chen, Z., Zhang, H., Xia, B., Wang, P., Jiang, T., Song, M., & Wu, J. (2013). Association of PTPN22 gene (rs2488457) polymorphism with ulcerative colitis and high levels of PTPN22 mRNA in ulcerative colitis.International Journal of Colorectal Disease,28(10), 1351–1358.https://doi.org/10.1007/s00384-013-1671-3[4]Machado-Contreras, J. R., Muñoz-Valle, J. F., Cruz, A., Salazar-Camarena, D. C., Marín- Rosales, M., & Palafox-Sánchez, C. A. (2016b). Distribution of PTPN22 polymorphismsin SLE from western Mexico: correlation with mRNA expression and disease activity.Clinical and Experimental Medicine,16(3), 399–406.https://doi.org/10.1007/s10238-015-0359-0Disclosure of Interests:None declared
Objective: Seclusion and restraint (S/R) are emergency safety measures to manage aggressive behaviour and prevent physical harm to self and others during psychiatric hospitalisations. Antipsychotics have been reported to reduce theincidence of S/R events during psychiatric hospitalisation. This study explores factors associated with inpatient S/R events and investigates whether long-acting injectable (LAI) antipsychotic prescription is associated with a reduction of S/R events.Method: Data on the number of S/R events during hospital stay were collected from the medical records of 741 psychiatric inpatients admitted between 2012 and 2017, and categorised into groups of 0, 1–2 and recurrent (≥3) S/R. Multinomial logistic regression analysis was performed to find the association between S/R events and several demographic and clinical variables, including the time to initiation of LAI (TLAI).Results: TLAI was not significantly associated with S/R events. Antipsychotic medication prescription was associated with a decreased risk of recurrent S/R events (OR = 0.47; 95% Cl = 0.24–0.92), however, it was not significant for the group having 1–2 S/R events (OR = 0.74; 95% Cl = 0.37–1.49). Individuals withrecurrent S/R events were more likely to have forensic admission, transfer from jail or supervised facility, higher psychiatric comorbidity, and higher inpatient medications and prescription changes.Conclusion: Early initiation of LAI antipsychotics was not associated with S/R events; however, routine oral antipsychotic medication prescription was associated with decreased risk of S/R events. Specific predictors of S/R episodesmay be used in preventative efforts aimed at decreasing S/R events.
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