Littérature scientifique sur le sujet « Realtime qPCR »

Produktivitas ikan budidaya dapat ditingkatkan melalui teknologi transgenesis. Populasi ikan lele transgenik cepat tumbuh telah dihasilkan dan karakter biologisnya telah diketahui. Namun informasi zigositas ikan lele transgenik perlu ditelaah lebih lanjut. Penelitian ini bertujuan untuk mengidentifikasi zigositas ikan lele transgenik F-2. Zigositas ikan lele transgenik diidentifikasi dengan menggunakan metode real-time qPCR (RT-qPCR) dan uji progeni. Identifikasi zigositas melalui uji progeni, dilakukan dengan mendeteksi transgen (PhGH) pada individu-individu F-3 hasil persilangan transgenik F-2 dengan non-transgenik. Hasil penelitian menunjukkan bahwa zigositas pada ikan lele transgenik F-2 dapat diidentifikasi dengan menggunakan metode RT-qPCR. Semua ikan transgenik F-2 adalah heterozigot, dengan nilai 2-Ct yang hampir sama tiap individu F-2, yaitu berkisar 0,80-0,99. Identifikasi zigositas dengan metode RT-qPCR menunjukkan hasil yang sama dengan uji progeni, semua transgenik F-2 tidak menghasilkan 100% anakan F-3 positif transgen. Pada uji progeni, transmisi transgen pada penelitian ini tidak mengikuti hukum segregasi Mendel, dengan kisaran sebesar 5%-40%.Fish farming productivity can be increased by transgenesis technology. On the previous study, transgenic African catfish population fast growing has been produced and its biological characters has been known. However information of transgenic zygosity of catfish should be examined. The aim of this study was to identify the zygosity of F-2 transgenic African catfish. The zygosity of F-2 transgenic was identified by real time-qPCR (RT-qPCR) method and progeny test. Further, identification of zygosity F-2 transgenic African catfish was confirmed by progeny test, while F-2 transgenic African catfish was mated with non-transgenic. Identification of zygosity F-2 transgenic was conducted by detection PhGH gene (transgene) in F-3 transgenic African catfish population. Transgene transmission was evaluated by PCR method. The result showed that the zygosity F-2 transgenic African catfish could be identified by RT-qPCR method. All F-2 transgenic African catfish were heterozygous, where as the 2-Ct value was almost same for all individual, which ranges from 0.80 to 0.99. The result of zygosity identification using RT-qPCR method was as same as that of progeny test. In the progeny test, transgene transmission in this study was non-Mendelian segregation, with ranges of 5%-40%.

Ae. aegypti and Ae. albopictus have an important role in DHF transmission because they can simultaneously transmit the dengue virus vertically / transovarially or horizontally. This phenomenon indicates the persistence of the dengue virus by vectors. The aim of this research was to prove the persistence of the transovarial-transgenerational dengue virus (DENV-1,2,3,4) with real time polymerase chain reaction (qPCR) in Ae. aegypti and Ae. albopictus (Diptera: Culicidae). Quasi experimental design with intervention infects DENV 1-2-3-4 serotypes in Ae. aegypti and Ae. albopictus intratoracally. Research population Ae. aegypti and Ae. albopictus laboratory colony females. Dengue virus detection uses real-time polymerase chain reaction (qPCR). Transovarial detection by qPCR indicates detection of dengue virus in Ae. albopictus DENV-1 to progeny 1 (F1), DENV-2 and DENV-3 to F2, DENV-4 to F3. Next to Ae. aegypti DENV-1 to 1st progeny (F1), DENV-2 to F2, DENV-3 to F4 and DENV-4 to F3. there was no difference in MIR value (p value: 0.356) for the four serotypes in Ae. albopictus and Ae. aegypti. DENV-3 is the most persistent serotype in Ae. aegypti with 83.3% MIR and DENV-4 were the most persistent serotypes in Ae.albopictus with 100% MIR. The need to improve vector control models that focus not only on the main vector, but also other co-vectors.

This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.

Preventive chemotherapy (PC), consisting of the regular distribution of anthelmintics to populations or groups of populations at risk, is the primary tool used to control soil-transmitted helminth (STH) infections. This strategy, whilst cost-effective, raises the concern of potential emergence of drug resistance. The efficacy of anthelmintics against STH infections is measured using cure rate (CR) and egg reduction rate (ERR), using microscopy-based techniques such as the Kato-Katz thick smear. However, Kato-Katz has low sensitivity, especially for low-intensity infections, and requires fresh samples that need to be processed quickly. Realtime quantitative PCR (qPCR), which is more sensitive, is emerging as a “gold standard” for STH diagnostics given its higher sensitivity (important in low prevalence settings) and ability to differentiate hookworm species, while sodium nitrate flotation (SNF) may provide a low-cost more sensitive and practical alternative to Kato-Katz in the field. In this study, we examined the efficacy of a locally manufactured brand of albendazole 400 mg (“Alzental”) against hookworm in Đắk Lắk province, Vietnam, using both qPCR and SNF. For qPCR, formulae to convert qPCR cycle threshold (Ct) values into eggs per gram of faeces (EPG) were utilised to determine efficacy calculations, and these values directly compared with efficacy values generated using SNF. Factors associated with CR and ERR were examined, and Alzental tablet quality was assessed by comparing with an Australian TGA-approved equivalent “Eskazole” tablet. We observed a CR and ERR of 64.9% and 87.5% respectively using qPCR, and 68.4% and 67.6% respectively using SNF. The tablet composition of Alzental was comparable to Eskazole in terms of active albendazole drug concentration with no evidence of impurities. This study demonstrates that the efficacy of Alzental against hookworm is within the range of previously reported studies for albendazole 400 mg. The study also demonstrates the value of qPCR and SNF as alternatives to standard Kato-Katz methodology for assessment of anthelmintic efficacy.

Two controlled environment experiments were performed to test the effects of nitrogen, phosphorus and carbohydrates on endophyte (Neotyphodium lolii) and alkaloid concentrations in ryegrass (Lolium perenne). Three perennial ryegrass cultivars ('high sugar grasses' AberDove and AberDart; control Fennema) that differ in carbohydrate content were infected with three strains of N. lolii (common strain, CS; AR1; AR37). Infected and uninfected plants were grown under high (9 mM) and low (2.25 mM) nitrogen (AberDove, Fennema; CS, AR1, AR37) or under high (2 mM KH2PO4) and low (0.05 mM KH2PO4) phosphorus (AberDart, Fennema; CS, AR1). Quantitative realtime Polymerase Chain Reaction (qPCR) was used to estimate endophyte concentrations in harvested leaf tissues. High N and P supply as well as high carbohydrate content of the host grass reduced endophyte concentrations. Alkaloid production was also reduced under both increased N supply and in the high sugar cultivar, and was linearly related to endophyte concentration (except ergovaline). The results stress the need for wider quantification of fungal endophytes in the grassland/ foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes, or increasing nutrient inputs, affect the role of endophytes in grassland ecosystems. Keywords: Neotyphodium lolii, foliar endophyte, Lolium perenne, perennial ryegrass, qPCR, high sugar ryegrass, nitrogen, phosphate, carbohydrate, AR1, AR37, alkaloids

Background Keratoconus (KTCN) is a progressive eye disease, characterized by changes in the shape and thickness of the cornea that results in loss of visual acuity. While numerous KTCN candidate genes have been identified, the genetic etiology of the disease remains undetermined. To further investigate and verify the contribution of particular genetic factors to KTCN, we assessed 45 candidate genes previously indicated as involved in KTCN etiology based on transcriptomic and genomic data. Methods The RealTime ready Custom Panel, covering 45 KTCN candidate genes and two reference transcripts, has been designed. Then, the expression profiles have been assessed using the RT-qPCR assay in six KTCN and six non-KTCN human corneas, obtained from individuals undergoing a penetrating keratoplasty procedure. Results In total, 35 genes exhibiting differential expression between KTCN and non-KTCN corneas have been identified. Among these genes were ones linked to the extracellular matrix formation, including collagen synthesis or the TGF-β, Hippo, and Wnt signaling pathways. The most downregulated transcripts in KTCN corneas were CTGF, TGFB3, ZNF469, COL5A2, SMAD7, and SPARC, while TGFBI and SLC4A11 were the most upregulated ones. Hierarchical clustering of expression profiles demonstrated almost clear separation between KTCN and non-KTCN corneas. The gene expression levels determined using RT-qPCR showed a strong correlation with previous RNA sequencing (RNA-Seq) results. Conclusions A strong correlation between RT-qPCR and earlier RNA-Seq data confirms the possible involvement of genes from collagen synthesis and the TGF-β, Hippo, and Wnt signaling pathways in KTCN etiology. Our data also revealed altered expression of several genes, such as LOX, SPARC, and ZNF469, in which single nucleotide variants have been frequently identified in KTCN. These findings further highlight the heterogeneous nature of KTCN.

ABFs play a key role in regulating plant osmotic stress. However, in Tartary buckwheat, data on the role of ABF genes in osmotic stress remain limited and its associated mechanism in osmoregulation remain nebulous. Herein, a novel ABF family in Tartary buckwheat, FtbZIP12, was cloned and characterized. FtbZIP12 is a transcriptional activator located in the nucleus; its expression is induced by NaCl, mannitol, and abscisic acid (ABA). Atopic expression of FtbZIP12 in Arabidopsis promoted seed germination, reduced damage to primary roots, and improved the tolerance of seedlings to osmotic stress. The quantitative realtime polymerase chain reaction (RT-qPCR) results showed that the expressions of the typical genes related to stress, the SOS pathway, and the proline synthesis pathway in Arabidopsis were significantly (p < 0.05) upregulated under osmotic stress. FtbZIP12 improved the osmotic pressure resistance by reducing the damage caused by reactive oxygen species to plants and maintained plant homeostasis by upregulating the expression of genes related to stress, osmotic regulation, and ion homeostasis. This study identified a key candidate gene for understanding the mechanism underlying osmotic-stress-regulated function in Tartary buckwheat, thereby providing a theoretical basis for improving its yield and quality.

Emerging evidence suggests that reactive oxygen (ROS) and nitrogen (RNS) species can contribute to diverse signalling pathways of inflammatory and tumour cells. Cucurbitacins are a group of highly oxygenated triterpenes. Many plants used in folk medicine to treat cancer have been found to contain cucurbitacins displaying potentially important anti-inflammatory actions. The current study was designed to investigate the anti-ROS and -RNS effects of cucurbitacin L 2-O-β-glucoside (CLG) and the role of these signaling factors in the apoptogenic effects of CLG on human colon cancer cells (HT-29). This natural cucurbitacin was isolated purely fromCitrullus lanatusvar.citroides(Cucurbitaceae). The results revealed that CLG was cytotoxic to HT-29. CLG increased significantly (P<0.05) RNA and protein levels of caspase-3 in HT-29 cells when verified using a colorimetric assay and realtime qPCR, respectively. The results showed that lipopolysaccharide/interferon-gamma (LPS/INF-γ) increased nitrous oxide (NO) production inR AW264.7macrophages, whereas N(G)-nitro-L-argininemethyl ester (L-NAME) and CLG curtailed it. This compound did not reveal any cytotoxicity on RAW264.7 macrophages and human normal liver cells (WRL-68) when tested using the MTT assay. Findings of ferric reducing antioxidant power (FRAP) and oxygen radical absorption capacity (ORAC) assays demonstrate the antioxidant properties of CLG. The apoptogenic property of CLG on HT-29 cells is thus related to inhibition of reactive nitrogen and oxygen reactive species and the triggering of caspase-3-regulated apoptosis.

Purpose : Ovarian cancer is the most lethal of gynecological malignancies. Recently, the development of microRNA (miRNA) -based therapeutics that could impact broad cellular programs, leading to inhibition of cancer cell viability, is gaining attention in the therapeutic landscape. The therapy is based on the presence of aberrant expressions of miRNA in cancer cells. Decreasing of tumor suppressor miRNA expression causes upregulation of oncoprotein, which worsens the prognosis of the ovarian cancer. Methods: miR-155-5p mimics were carried by chitosan nanoparticles using new nanotechnology methods. Cellular uptake of miRNA was assessed by fluorescence microscope while MTT and qPCR assay were used to determine miRNA profile and the effect of CS-NP/miRNA on SKOV3 cells. Results: Results of profiling validated using quantitative realtime-polymerase chain reaction (PCR) found one of the most altered tumor suppressor miRNAs, miR-155-5p was downregulated 892.15-fold. According to bioinformatic analysis we identified the miRNA could recognize and regulate HIF1α expression. Transfection of mimics for miR-155-5p showed significantly increased miR-155-5p endogen SKOV3 expression level compared to the control group. We found differences after transfection mimics for miR-155-5p 31.5 and 63 nanoMolar. Increasing of miR-155-5p endogen lead to diminished SKOV3 viability (by 30%; <0.05 at concentration 80 nanoMolar). These mimics may cause an increase in upregulated miR-155-5p endogen that can reduce HIF1α expression. Here we found 2-fold and 2.8-fold reduction of HIF1α expression level after transfection compared to the control group. Conclusion: According to these findings, the mimics miR-155-5p can inhibit ovarian cancer cell proliferation by regulating HIF1α expression.

Background/Aims: Increasing evidence indicates that Huaier extract has promising therapeutic effects against cancer. However, the mechanisms that underlie its anti-tumor effects remain unclear. In recent years, various studies have shown that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cancer development and progression. Here, we explored the role of lncRNAs in Huaier-induced tumor suppression. Methods: Microarray profiling was performed to identify the candidate lncRNAs affected by Huaier extract. Quantitative realtime PCR (qPCR) was used to evaluate the transfection efficiency and the influence of Huaier extract on H19 expression. The effect of Huaier extract on the cell viability was examined by MTT. Moreover, the rates of apoptotic cells were detected using flow-cytometric analysis. Western blot analysis was applied to show the protein levels of CBL. Results: Microarray data derived from Huaier-treated breast cancer cells identified H19 as a potential target. Huaier extract reduced the expression of H19. The over-expression of H19 inhibited the cytotoxic effects of Huaier extract; in contrast, reduced H19 expression enhanced the function of Huaier extract. MiR-675-5p was identified as a mature product of H19. Moreover, Huaier extract reduced the miR-675-5p expression. Upregulating miR-675-5p reversed the inhibitory effects of Huaier extract, whereas downregulating miR-675-5p sensitized breast cancer cells to the effect of Huaier extract. In addition, Huaier extract increased the expression of CBL protein, a direct target of miR-675-5p. Conclusion: Collectively, the data demonstrate that Huaier extract reduces viability and induces apoptosis in breast cancer cells via H19-miR-675-5p-CBL axis regulation.

Atherosclerosis (also known as Arteriosclerotic Vascular Disease or ASVD) is the condition in which an artery wall thickens as the result of a build-up of fatty materials such as cholesterol. It is a syndrome affecting arterial blood vessels, a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by Low-density lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a hardening or furring of the arteries. It is caused by the formation of multiple plaques within the arteries. Other arteriosclerotic disease is aortic aneurysm usually affecting the abdominal tract of the vessel. The prevalence of abdominal aortic aneurysm (AAA) varies ranging from 4.1% to 11.5% in European men. This disorder is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and eventual aortic rupture. Rupture of AAA is responsible for 1.5% of the total mortality in males over 55 years of age. Both disease seem to share common causes in alteration in lipid metabolism and immune response against host and oxidised lipids, leading to a condition of chronic inflammation affecting all the vascular system, especially the big diameter arteries. There are several factors such as smoking, hypertension, hypercholesterolemia and male sex, which are well known risk factors for the development of AAA and carotid stenosis. However, better understanding of molecular mechanisms is an important step toward clarification of the pathophysiology, identification of genetic and molecular biomarkers and development of new therapeutic strategies for AAA and atherosclerosis in general. Gene expression profiling studies by microarray technologies are particularly appropriate to investigate and create working hypotheses to understand the pathophysiology of complex genetic tracts such as arteriosclerotic diseases. Previous microarray studies focused on AAA utilized RNA derived from aortic tissue samples. The use of tissue samples has several disadvantages, including the difficulty of obtaining control samples and bias introduced by use of normal specimens from non-age-matched cadavers, organ-transplant donors or patients with different diseases. Peripheral blood is a complex fluid with a high cellular turnover rate that provides physiological connectivity between tissues. Environmental or disease perturbations in the body may leave molecular signatures detectable by analyzing blood-derived RNA. Most importantly, since blood samples can be obtained readily and with little discomfort to patients, biomarkers derived from blood RNA provide an easier integration to clinical and imaging data for the diagnosis and prognosis of AAA and carotid stenosis. In this study we investigate the gene expression profile of venous whole peripheral blood obtained from AAA patients by using microarray technology to provide insight into systemic pathophysiological processes involved in this disease. Using the data obtained from the microarray analysis from which we detected 91 genes differentially expressed from patients and positive controls, we selected 10 gene belonging to pathways involved in lipid metabolism and immune response to deep our analysis and eventually confirm the microarray data in order to set up new reliable parameters for arteriosclerosis diseases diagnosis and prognosis. The genes we chosen are Lipid Metabolism: 8. Monoglyceride lipase - MGLL - U67963 - 11343 9. Free fatty acid receptor 2 - FFAR2 - AF024690 – 2867 10. Adiponectin receptor 1 - ADIPOR1 - AK001484 – 51094 11. Phospholipase A2, group IB (pancreas) - PLA2G1B - M21054 – 5319 12. Hydroxysteroid (17-beta) dehydrogenase 14 - HSD17B14 - NM_016246 3738 13. Acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain - ACADS Z80345 – 682 14. Low density lipoprotein receptor-related protein 5 - LRP5 - AF077820 - 4041 Immune system: 4. Toll-like receptor adaptor molecule 1 - TICAM1 - AF070530 - 7746 5. Interleukin 1 receptor accessory protein-like 1 - IL1RAPL1 - AJ243874 11141 6. Tumor necrosis factor type 1 receptor-associated protein - TRAP1 NM_016292 – 10131 We designed 10 couples of primers to analyze the expression levels of the genes onto 3 groups of subjects: 1. Control: N=40 2. AAA patients: N=40 3. Patients affected from carotid artery stenosis: N=40 We first analyze the expression levels of the control gene GAPDH of each subject and than we check the expression levels of the all ten genes comparing the resulting CT values with the ones gotten from the housekeeping gene. The result is the so-called ∆CT and we got these values for all the 120 subjects for everyone of the 10 genes in duplicates for a total of 1200 ∆CT values. Than we compare the average ∆CT values of the 80 patients (40 AAA + 40 carotid stenosis) with the control values to get the ∆∆CT values from which calculate the fold number (2-∆∆CT =FOLD) that provides a numeric but qualitative value of the expression level of the target gene that allow to compare the values with others present in literature. Our analysis are only semiquantitative for the moment but we first needed to check the reliability of the experimental approach. For the future studies we are going to use Taqman® probes to rise the specificity and sensitivity of the analysis, introducing a quantitative method using titolation commercial kit that allow to quantify the exact numbers of copies of an m-RNA transcript calculating the exact amount of m-RNA by substituting the CT values in a linear equation. We confirmed the microarray data and extended the results to the carotid stenosis patients. We were able to confirm the genes upregulation and hypothesise that PBMCs could be a suitable base for the detection of novel risk and prognostic indicators for the arteriosclerotic diseases. In fact our aim is to identify new easy-to-detect markers to predict the risk of developing an artery stenosis or most important, an aneurismatic lesion that is symptomless until the very late phases and whose success in treating is proportional to well-timed diagnosis. In the end seems that the right direction to look into are the metabolism of lipids and phospholipids, that are involved in eicosanoids production and degradation (via fatty acid metabolism) and cell signalling in the immune system.