Maryam M. Matin; David P. Hornby
Abstract
We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library ...
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We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.
Roghaye Lorestani; Sohrab Boozarpour; Sakineh Alijanpour; Leila Ahangar
Abstract
Schizophrenia is an irritating mental disorder that affects around 1% of the world's population. The immune system contributes to the onset of the disease, particularly through production and secretion of some cytokines. In patients with schizophrenia, the balance of Th1 to Th2 ratio ...
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Schizophrenia is an irritating mental disorder that affects around 1% of the world's population. The immune system contributes to the onset of the disease, particularly through production and secretion of some cytokines. In patients with schizophrenia, the balance of Th1 to Th2 ratio is often altered. In the present study, we investigated these changes by measuring the gene expression levels of IFN-γ and T-bet as Th1 indicators, as well as IL-4 and GATA-3 as representatives for Th2. Blood samples of schizophrenic patients (n=25) and healthy individuals (n=10) were obtained. Total RNA was extracted from leukocytes and cDNA synthesis was performed based on provided protocols. Real-time PCR technique was utilized for the assessment of gene expression levels. Results indicated a significant increase in the expression of IFN-γ and its transcription factor, T-bet, while IL-4 gene expression was reduced significantly. The expression level of GATA-3 gene revealed no meaningful changes. Altogether, results confirmed the relative shift of Th1 to Th2 status in the patient with schizophrenia and re-emphasize the importance of the inflammatory events in the incidence of the disease. Moreover, a new index was introduced based on the IFN-γ and T-bet genes expression, which can determine healthy condition with total accuracy of 79%.
Sepideh sadat Hosseini; Shadi Mehrzad; Halimeh Hassanzadeh; Hamid Reza Bidkhori; Mahdi Mirahmadi; Madjid Momeni-Moghaddam; Fatemeh Sadeghifar; Moein Farshchian
Abstract
Mesenchymal stem/stromal cells (MSCs) as one of the most important types of adult stem cells secrete a variety of immunomodulatory cytokines. However, their immunomodulatory features strongly depend on the molecular cross-talk between cells and the surrounding microenvironment. Hence, ...
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Mesenchymal stem/stromal cells (MSCs) as one of the most important types of adult stem cells secrete a variety of immunomodulatory cytokines. However, their immunomodulatory features strongly depend on the molecular cross-talk between cells and the surrounding microenvironment. Hence, some strategies were proposed to empower their beneficial effects during cell-therapeutic procedures to avoid confusing results. Licensing the cells with chemical compounds could be considered as one of the most applicable methods for induction of anti-inflammatory status in the cells. Human chorionic gonadotropin (hCG) is a pregnancy related hormone which has been shown to be essential for the establishment of a successful pregnancy. HCG supports the implantation of fetus in the maternal endometrium, due to its immunomodulatory effects. Moreover, the regulatory role of hCG has been previously mentioned in case of some autoimmune-based diseases. In the present study, the capacity of this hormone for induction of different immune-encountered genes expression was examined in primary cultures of human adipose tissue derived mesenchymal stem cells (Ad-MSCs). In this regard, Ad-MSCs were exposed to 10 IU of hCG for 72 hours. Molecular studies via quantitative Real-time PCR (qRT-PCR) experiments were performed to detect gene expression modifications based on the application of SYBR Green as the fluorescent dye and in comparison to the RPLP0 as the housekeeping gene. Results confirmed that hCG significantly upregulated TSG-6, TGF-β1, IL-1β and IL-6 expression levels comparing with the control group, while it downregulates COX-2 expression, and had no statistically significant effects on IL-10 andTDO2. In conclusion, priming Ad-MSCs with hCG may enhance the proliferation and immunoregulatory potential of these cells, although it needs further investigations to reveal involved molecular pathways.