Time PCR Technique Research Articles

Comparative Analysis of ITGΒ6 Gene Expression in Oral Cancer

The goal of this study was to determine the quality and quantity of ITGβ6 gene expression in oral squamous cell carcinoma (OSCC). Two Cell lines (H357) and (Vβ6) were used to analyses β6protein and gene expression by using BAC assay, SDS-PAGE gel, and western blotting, agarose gel as well as real- time PCR techniques. The western blotting and real-time PCR studies demonstrated high level of protein expressions in the transected Vβ6 cells line which shown as clear thick band. On the other hand, a protein expression of H357 cells line was less and gives weak band. Therefore, Squamous cell lines from the oral mucosa that express β6 are linked to increased invasion and progression. This highlights the significance of understanding cancer progression mechanisms and the impact of varying expression levels in different tumors.

Examining the Synergic Effect of Exosomes Derived from Mouse Mesenchymal Stem Cells and Low-frequency Electromagnetic Field on Chondrogenic Differentiation

Background: Cartilage has intrinsically limited healing power, and regeneration of cartilage damages has remained a challenge. Secreted products of mesenchymal stem cells have shown a new therapeutic strategies for cartilage injuries. Also it has been observed that low frequency electromagnetic field plays a key role in biological processes. Objective: This research was performed to investigate the synergic effect of mesenchymal stem cell-derived exosomes and low frequency electromagnetic field on chondrogenic differentiation. Methods: In this in vitro study, mesenchymal stem cell-derived exosomes were identified using AFM, SEM, TEM microscopy, and DLS method. Cells were treated in chondrogenic medium by exosomes, low frequency electromagnetic field, and the synergy of both. The cell survival was examined using MTT and Annexin methods, and cartilage differentiation was confirmed by Alcian blue staining. The expression of Sox-9, Acan, Col 2a1 and Col 10a1 genes was examined via Real- Time PCR technique on day 14 post-treatment. Results: The results confirmed the presence of exosomes with an approximate size of less than 100 nm. The results of Alcian blue revealed greater expression of glycosaminoglycans in the synergic treatment group compared to the other groups. Real-time PCR showed a significant increase in the expression of Sox-9, Acan, and Col 2a1 genes, as well as a significant reduction of Col 10a1 gene expression in the synergic treatment group compared to other groups. Conclusion: This study indicated that the synergic effect of exosome and low-frequency electromagnetic fields would lead to enhanced chondrogenic differentiation, which can be further explored in future clinical studies

The effect of BKV reactivation on cytokines behavior in kidney transplanted patients

BackgroundBK virus associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplanted recipients (KTRs). The current treatment for BKV nephropathy is decreasing the immunosuppressive regimen in KTRs. Interleukin-27 (IL-27) is a multifunctional cytokine that might be the front-runner of an important pathway in this regard. Therefore, in current study it is tried to evaluate the changes in the expression level of IL-27 and some related molecules, resulting from BKV reactivation in KTR patients.MethodsEDTA-treated blood samples were collected from all participants. Patients were divided into two groups, 31 kidney transplant recipients with active and 32 inactive BKV infection, after being monitored by Real time PCR (Taq-Man) in plasma. Total of 30 normal individuals were considered as healthy control group. Real time PCR (SYBR Green) technique is used to determine the expression level of studied genes.ResultsThe results of gene expression comparisons showed that the expression level of IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 genes was significantly higher in inactive group in comparison to active group. The expression level of TLR4 was lower in both active and inactive groups in comparison to control group. ROC curve analysis showed that IL-27 and IRF7 are significantly different amongst other studied genes. Finally, the analyses revealed that the expression level of most of the studied genes (except for TNF-α and TLR4) have significant correlation with viral load.ConclusionsOur findings revealed that IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 expression level is higher in inactive group and TLR4 expression level is lower in patients’ groups in comparison to control group. Also, ROC curve analysis showed IL-27 and IRF7 can significantly differentiate studied groups (BKV active vs. inactive). Therefore, these results might help elucidating the pattern in charge of BKV reactivation in kidney transplanted patients.

Auto-qPCR; a python-based web app for automated and reproducible analysis of qPCR data

Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app (https://auto-q-pcr.com/) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

Investigation of olfactory receptor family 51 subfamily j member 1 (OR51J1) gene susceptibility as a potential breast cancer-associated biomarker.

Among cancer treatment methods, targeted therapy using cancer-associated biomarkers has minimum side effects. Recently olfactory receptor (OR) family attracts the researcher’s attention as a favorable biomarker of cancer. Here, a statistical approach using complete data from the human protein atlas database was used to evaluate the potential of OR51J1 gene as a cancer-associated biomarker. To confirm the findings of statistical analysis, the OR51J1 mRNA and protein expression levels in breast tumor and normal tissue were measured using quantitative Real Time PCR (qRT-PCR) and immunohistochemistry (IHC) techniques. The association with clinicopathological factors was analyzed. Statistical analysis revealed that OR51J1 has a high expression level in more than 20 types of cancer tissues without any expression in 44 normal tissues. In 15 cancer types, including breast cancer, expression score was more than 90%. The qRT-PCR analysis in breast cancer showed OR51J1 have significantly higher expression level in tumors than normal tissues (2.91 fold). The IHC results showed OR51J1 expression on other cellular subtypes than tumor and normal cells, including myoepithelium, fibroblast, and lymphocytes. OR51J1 protein expression in invasive cells, as well as its overall score, showed a significant correlation with ER and PR expression and breast cancer (BC) subtypes. Results revealed the potential of OR51J1 as a cancer-associated biomarker for the diagnosis of breast cancer at the mRNA level.

Molecular Gene Expression of Toll-Like Receptors 4 & 10 in Cellular Subsets of Human Peripheral Blood among Patients with Prostatitis: Conventional, Real Time Pcr and DNA Sequencing Techniques

The Aim of this study was to determine Immunogenetic expression of Toll-like receptor gene clusters related to prostatitis, to give acknowledge about Role of TLR in prostatitis immunity in men from Basrah and Maysan provinces. A case–control study included 135 confirmed prostatitis patients And 50 persons as a control group. Data about age, marital status, working, infertility, family history and personal information like (Infection, Allergy, Steroid therapy, Residency, Smoking, Alcohol Drinking, Blood group, Body max index (BMI) and the clinical finding for all patients of Prostatitis were collected , The molecular expression study include extracting DNA from blood of Prostatitis patients , Prostitis patients and Control group by using specific primers for conventional PCR and Real Time PCR , the amplification of all extracted DNA from blood samples was preform and confirm by using electrophoresis with (100volt/30min). The amplification of all extracted DNA from blood samples was preform and confirm by using electrophoresis with (100volt/30min) , the result of this estimation revealed that the amplified DNA(PCR product) was 227bp for TLR4 on agarose gel (1%) , (50voltage for 1hour ) with a presence 100% , (PCR product) was 279bp for TLR10 on agarose gel(1%) , (50volt/1hour) with a presence 80%. The results of the present study indicate that the Toll like receptor alleles associated with risk of prostatitis.

Introduction of telomerase reverse transcriptase gene into epidermal stem cells derived from human skins

Telomerase extends the proliferation and prevent replicative senescence in most somatic cells. Whether it has similar function in human epidermal stem cells remains to be determined. In this study, the human telomerase reverse transcriptase (hTERT) cytalytic subunit was introduced into epidermal stem cells derived from human fetal skins. The expression of hTERT mRNA, protein and telomerase activity in the transduced cells was observed by real time PCR (RT-PCR) techniques, western blot analysis and telomeric repeat amplification protocol enzyme-linked immunosorbent assay (TRAP-ELISA) assay, respectively. The proliferation of the transduced cells was examined. The results showed the introduction of hTERT into the epidermal stem cells upregulated the expression of the hTERT gene and protein. And also the hTERT-transduced epidermal stem cells exhibited significantly elevated telomerase activity and proliferation. These works establish the base of further gene modification, monoclone screening and cell differentiation mechanism. The ability to maintain the biological characteristics and proliferation of epidermal stem cells could have important applications in wound healing and skin tissue engineering.

Quantitative Detection of <i>Helicobacter pylori</i> by Real Time PCR in Drinking Water—Environmental and Public Health Risk Significance

Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms; nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panama (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriqui were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized; also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori; removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panama it was determined that 86% of the samples were found positive for the presence of H. pylori; removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.

Isolation of Enterococcus Faecium and Enterococcus Cecorum from Bovine Rumen Using Modern Techniques

This study aimed to isolate Enterococcus sp. from bovine rumen samples. Isolation were carried out using classical method by using Streptococcus selective medium then the isolates were identificated using modern techniques by real time PCR, gel electrophoresis and DNA sequencing techniques. Results showed that Enterococcus sp. enumerated in the range from 103 to 105 CFU/mL. One of the isolates identified as Enterococcus faecium and two other isolates were Enterococcus cecorum.

Real-Time PCR for Detection CP1 & CP5 Virulence Factors of Entamoeba Histolytica in Patients Stool Samples in Al - Najaf Al- Ashraf Province

Objective: It aims to determine the genes characterization of CP1 and CP5 pathogenic factors of E.histolytica parasitic in stool samples of patients in AL- Najaf Al Ashraf Province Methodology: This study was carried out for 6 months in the first of December 2012 till the end of June 2013, The samples collected of Al-Sadar teaching hospital, AL-Zahra maternity and children hospital, AL-abasia hospital, AL-Forat hospital, AL-Hakime hospital, AL-Manathera hospital and private clinic in Al- Najaf governorate. Results: To detect the major virulence factors (V.F.) cysteine proteinase1 (Cp1), cysteine proteinase 5 (CP5) of E.histolytica, Real -Time PCR technique was conducted, by using specific primers for E.histolytica, the results showed that 40 samples out of 162 were positive, out of these positive samples, there were 16 samples positive for C P1, 24 samples were positive for CP5. Conclusion: CP1 & CP5 is one of the most important virulence factors in E.histolytica such as toxin like activity. Recommendation: further study development of molecular diagnosis such as real time PCR for other non-pathogenic Entamoeba species which found in human, such as E.moskoviskii in comparison with E.histolytica, because the RT-PCR quantitative and qualitative improved method for the specific diagnosis of E.histolytica infection.

KRAS aKtive, an Italian network for assessment of KRAS mutations in colorectal cancer patients: Results on 7,432 cases.

e14042 Background: The KRAS aKtive program was started on March 2009, promoted by the Italian Association of Medical Oncology (AIOM) and the Italian Society of Surgical Pathology and Cytopathologyy (SIAPEC) to support the activity of oncologists and pathologists involved in the management of metastatic colorectal cancer patients who need the assessment of the mutational status of the KRAS gene. Methods: The program was specifically devised to facilitate the exchange of biologic material, clinicopathological data and diagnostic reports within a network of oncologist, pathologists and pathology/molecular biology reference laboratories throughout Italy, connected through the site www.kras-aKtive.it. KRAS mutation analysis was performed by Sanger sequencing (SS), real time PCR or other techniques, including pyrosequencing and hybridization strip assays. Data were collected in a common database. Results: The KRAS aKtive program has involved 478 oncologists, 144 pathologists, and 24 reference laboratories. A total of 7,432 KRAS mutations analyses were performed. The tests were informative in 7,265 cases (98%). The vast majority of tests (5,626 cases, 77.4%) were conducted by SS. In 529 (7.3%) cases a real-time PCR assay was used, other detection techniques were used in 1,110 (15.3%) cases. KRAS mutations at codons 12-13 were detected in 2,874 cases (39,6%). The frequency of mutations detected by real-time PCR or other techniques (45%, and 43%, respectively) was significantly higher (p=0.002, and p=0.008%, respectively) than that observed by SS (38%). The percentage of cases evaluated by non-SS-based methods has increased during the first three years of the program. Conclusions: The results of this large survey allow an accurate estimation of the actual prevalence of KRAS mutations and their types in caucasian colorectal cancer patients. Our data indicate that the frequency of mutations detected by non-SS-based methods is higher than that obtained by SS.

DMP1 and DSP localization during in vitro odontoblast like cell differentiation

The aim of this study was to analyze the temporal and spatial localization of dentin matrix protein 1 (DMP1) and dentin sialoprotein (DSP) in human dental pulp cells differentiated in vitro towards odontoblast-like cells. Following to a notable injury to the tooth, the odontoblasts die and a new generation of odontoblast-like cells differentiate from pulp progenitor cells [1]. They are responsible for the secretion of the reparative dentin matrix. In this study, morphological changes and the analysis of DMP1 and DSP were investigated starting from human dental pulp cells differentiated in vitro. Alizarin red staining, transmission and scanning electron microscopy in combination with Real Time PCR and immunofluorescence techniques were performed to demonstrate the odontoblast-differentiated phenotype and the specific expression and localization of DMP1 and DSP. The results showed a gradual calcium deposition to demonstrate a mineralization matrix. Electron microscopy analysis showed fibroblast-like cells characterized by a large number of mitochondria and a developed rough endoplasmic reticulum and Golgi apparatus involved in intense protein synthesis. A large extracellular matrix was detectable in which collagen type I fibers were detected. The DMP1 and DSP showed low expression and nuclear localization at the beginning of the odontoblast-like cell differentiation. After 21 days, elevated cytoplasm expression was detectable for both odontogenic markers.