Podcasts about pcr qpcr

Polymerase chain reaction (PCR) was discovered in 1983 by Kary Mullis and Michael Smith, who were jointly awarded the Nobel Prize in Chemistry in 1993. Since then, PCR has been a cornerstone method that has been a pillar of discovery and applied science. The various types of PCR are sometimes confusing, and the relative pros and cons of each method are not always clear, which is why it's so great to have this episode's guest explain them all in a simple and clear-cut way. Dave Bauer, PhD, is an Application Scientist at Thermo Fisher Scientific that specializes in real time PCR (qPCR) and digital PCR (dPCR).  He has an educational background in physics, mathematics, and biology, but what's more important is that Dave loves to help others learn and to break down a topic's complexities to make it more understandable and approachable.  In this episode we hear Dave explain the difference between qPCR and dPCR, the importance of Poisson statistics to dPCR, dead volume, reaction chamber volume consistency, and more.  We learn how qPCR and dPCR complement each other and how they relate to sequencing methods for applications like single nucleotide polymorphism (SNP) detection.  As you've come to expect from Absolute Gene-ius, you also get a good sense of who Dave is and how he got to his current role. We learn about how he knew right away that academia wasn't for him, how he ended up unexpectedly working in forensics after his PhD, and how he eventually landed in his current Application Scientist role. Dave shares some great insights and advice, including how students should care less about their degree's name and more about what techniques they're learning and using in their studies. Visit the Absolute Gene-ius page to learn more about the guest, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.This episode includes the following sound effects from freesound.org, licensed under CC BY-ND 4.0:“Sax Jazz,” by alonart“Balloon Pop / Christmas cracker / Confetti Cannon,” by Breviceps“Crowd Cheering,” by SoundsExciting

Bioinformatics is a relatively new field of science that is very interdisciplinary in nature. Its practitioners use a mixture of biology, chemistry, physics, statistics, and computer science to develop methods and software aimed at helping integrate and understand biological and other data.  Our guest for this episode is Nikhil Ram Mohan, Staff Scientist at the Stanford University School of Medicine. He describes bioinformatics as the bridge to understanding biology. We learn about his international studies and path that brought him to this current role and field of study, and then dive into some of his recent work. Here he and his team analyze biobank samples using digital PCR (dPCR) and quantitative PCR (qPCR) and compare results from the two while correlating results with additional data available for each sample to determine if SARS-CoV-2 RNA detection and quantification in blood can serve to help predict potential for patient coinfection. Their work found that dPCR was able to detect SARS-CoV-2 in samples that were negative when evaluated by qPCR and that a series of biomarkers can help predict coinfection.   We also get to hear a bit of Nikhil's interesting personal story, which includes his undergraduate engineering studies in India and leaving his native country for the first time when he moved to the U.S. for graduate school.  We learn how he managed changes in culture, what he loves about teaching, and about him being a new father.  Visit the Absolute Gene-ius page to learn more about the guest, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System. 

Visit the Absolute Gene-ius page to learn more about the guest, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.The details of what make digital PCR (dPCR) different from real-time, or quantitative PCR (qPCR) are relatively simple but not always explained very well. Likewise, it's not always clear which use cases are a good fit for dPCR, and which others simply don't require the power of dPCR. The power of digital PCR is real, if you understand it.In this episode we enlist Marcia Slater, a self-described “PCR guru” to explain digital PCR and its power. She covers the basic differences between dPCR and qPCR and then delves into the details of where dPCR derives its power and where it shines. With over 20 years' experience in helping customers troubleshoot PCR, Marcia makes is easy to understand key terms and concepts related to dPCR, including:Sub-reactionsPoisson statisticsStatistical power and confidence intervalsControls and false negatives vs. true negativesDead volumeDynamic rangeMultiplexingMarcia also covers some great examples of where the absolute quantification of dPCR is a great fit and how it's even used to qualify and quantify standards for qPCR. Multiplexing and how its used to do molecular integrity evaluations for gene therapy applications is also discussed.As always with the Gene-ius series, you'll also get to learn about more than Marcia's science chops. We learn about her unlikely career path from growing up on a livestock farm to her storied role in helping produce “data so beautiful it should be framed.” We even get into her rediscovered love of raising animals, including her beloved panda alpaca with a name you cannot forget!

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.29.518315v1?rss=1 Authors: Frorup, C., Gerwig, R., Sondergaard Svane, C. A., Mendes Lopes de Melo, J., Floyel, T., Pociot, F., Kaur, S., Storling, J. Abstract: ObjectiveEndoC-{beta}H5 is a newly established human beta-cell model which may be superior to previous models of native human beta cells. Exposure of beta cells to proinflammatory cytokines is a widely used in vitro model of immune-mediated beta-cell failure in type 1 diabetes and we therefore performed an in-depth characterisation of the effects of cytokines on EndoC-{beta}H5 cells. MethodsThe sensitivity profile of EndoC-{beta}H5 cells to the toxic effects of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), interferon {gamma} (IFN{gamma}) and tumour necrosis factor- (TNF) was examined in titration and time-course experiments. Cell death was evaluated by caspase 3/7 activity, cytotoxicity, viability, TUNEL assay and immunoblotting. Mitochondrial function was evaluated by extracellular flux technology. Activation of signalling pathways and major histocompatibility complex (MHC) class I expression were examined by immunoblotting, immunofluorescence, and real-time quantitative PCR (qPCR). Glucose-stimulated insulin secretion (GSIS) and cytokine-induced chemokine secretion were measured by ELISA and Meso Scale Discovery multiplexing electrochemiluminescence, respectively. Global gene expression was characterised by stranded RNA sequencing. ResultsCytokines increased caspase activity and cytotoxicity in EndoC-{beta}H5 cells in a time- and dose-dependent manner. The proapoptotic effect of cytokines was primarily driven by IFN{gamma}. Cytokine exposure caused impaired mitochondrial function, diminished GSIS, and induced secretion of chemokines. At the signalling level, cytokines increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1) but not c-jun N-terminal kinase (JNK) and did not cause degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I{kappa}B). MHC class I was induced by cytokines. Cytokine exposure caused significant changes to the EndoC-{beta}H5 transcriptome including upregulation of HLA genes, endoplasmic reticulum stress markers, and non-coding RNAs. Among the differentially expressed genes were several type 1 diabetes risk genes. ConclusionsOur study provides detailed insight into the functional and transcriptomic effects of cytokines on EndoC-{beta}H5 cells. This knowledge will be helpful for future investigations studying cytokine effects in this cell model. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Identification of variant infectious bronchitis viruses (IBVs) has improved with the development of a real-time PCR (qPCR) test that can distinguish the type of virus and determine if it’s vaccine- or field-related.

I denne podcast fortæller Katrine og Kim om qPCR. Hvad er forskellen på PCR og qPCR, hvad er farver, hvad kan man bruge det til og meget mere. Få svar på det hele ved at lytte til denne podcast

The mitochondria of non-bilaterian metazoans display a staggering diversity of genome organizations and also a slow rate of mtDNA evolution, unlike bilaterians, which may hold a key to understand the early evolution of the animal mitochondrion. Octocorals are unique members of Phylum Cnidaria, harboring several atypical mitochondrial genomic features, including a paucity of tRNA genes, various genome arrangements and the presence of novel putative mismatch repair gene (mtMutS) with various potential biological roles. Thus octocorals represents an interesting model for the study of mitochondrial biology and evolution. However, besides its utility in molecular phylogenetics, the mtDNA of octocorals is not studied from the perspective of DNA repair, oxidative stress response or gene expression; and there is a general lack of knowledge on the DNA repair capabilities and role of the mtMutS gene, response to climate-change, and mtDNA transcription in absence of interspersed tRNA genes of octocoral mitochondrial genome. In order to put the observed novelties in the octocoral mitochondria in an evolutionary and an environmental context, and to understand their potential functions and the consequences of their presence in conferring fitness during climate change induced stress, this study was undertaken. This dissertation aims to explore the uniqueness and diversity of octocoral mtDNA from an environmental as well as an evolutionary perspective. The thesis comprises five chapters exploring various facets of octocoral biology. The introductory section provides basic information and elaborates on the importance of studying non-bilaterian mitochondria. The first chapter sets the base for subsequent gene expression studies. Octocorals are extensively studied from a taxonomic and phylogenetic point of view. However, gene expression studies on these organisms have only recently started to appear. To successfully employ the most commonly used gene expression profiling technique i.e., the quantitation real-time PCR (qPCR), it is necessary to have an experimentally validated, treatment-specific set of stably expressed reference genes that will support for the accurate quantification of changes in expression of genes of interest. Hence, seven housekeeping genes, known to exhibit constitutive expression, were investigated for expression stability during simulated climate-changed (i.e. thermal and low-pH) induced stress. These genes were validated and subsequently used in gene expression studies on Sinularia cf. cruciata, our model octocoral. The occurrence of a mismatch repair gene, and the slow rates of mtDNA evolution in octocoral mitogenome calls for further investigations on the potential robustness of octocoral mitochondria to the increased oxidative stress. The second chapter presents a mitochondrion-centric view of climate-change stress response by investigating mtDNA damage, repair, and copy number dynamics during stress. The changes in gene expression of a set of stress-related nuclear, and mitochondrial genes in octocorals were also monitored. A robust response of octocoral mitochondria to oxidative mtDNA damage was observed, exhibiting a rapid recovery of the damaged mtDNA. The stress-specific regulation of the mtMutS gene was detected, indicating its potential involvement in stress response. The results highlight the resilience potential of octocoral mitochondria, and its adaptive benefits in changing oceans. The tRNA genes in animal mitochondria play a pivotal role in mt-mRNA processing and maturation. The influence of paucity of tRNA genes on transcription of the mitogenome in octocorals has not been investigated. The third chapter steps in the direction to understand the mitogenome transcription by investigating the nature of mature mRNAs. Several novel features not present in a “typical” animal mt-mRNAs were detected. The majority of the mitochondrial transcripts were observed as polycistronic units (i.e. the mRNA carrying information for the synthesis of more than one protein). 5’ and 3’ untranslated regions were delineated for most protein-coding genes. Alternative polyadenylation (APA) of mtMutS gene and long non-coding RNA (lncRNA) for ATP6 were detected and are reported for the first time in non-bilaterian metazoans providing a glimpse into the complexity and uniqueness of mtDNA transcription in octocorals. The mismatch repair (MMR) mechanism plays a crucial role in mutation avoidance and maintenance of genomic integrity. Its occurrence in animal mitochondria remains equivocal. Octocorals are the only known animals to posses an mtDNA-encoded MMR gene, the mtMutS, speculated to have self-contained DNA repair capability. In order to gain knowledge of the MMR activity in the octocoral mitochondria MMR assays using the octocoral mitochondrial fraction is necessary. A prerequisite for this assay is the availability of an MMR-substrate, which is a DNA fragment, usually a plasmid, containing the desired mismatch lesion (i.e. a heteroduplex) and a nicked strand. However, the methods to prepare such a substrate are time consuming and technically demanding. Chapter four describes two convenient and flexible strategies that can be used in parallel to prepare heteroduplex MMR substrate using a common plasmid and routine molecular biology techniques. This method should aid in MMR investigations in general, helping to advance this field of research. The mtMutS gene mentioned above is a bacterial homolog, predicted to have been horizontally transferred to the octocoral mitogenome. However, unlike the bacterial mutS, which is extensively studied, protein expression studies of the octocoral mtMutS gene are lacking. To investigate the biological role of the mtMutS protein, in vitro, and to gain knowledge on its structure and function, the expression of the gene in a bacterial host is necessary. The fifth chapter discusses the characteristics of the mtMutS protein, the efforts to express it in E. coli and some necessary precautions to be taken while working with the expression of such mtDNA-encoded proteins for the research in future. This dissertation elucidates and contributes to the understanding of the unexplored complexity of non-bilaterian mitochondria. It deals for the first time with DNA repair, gene expression and gene function, encompassing an integrative analysis of DNA, RNA and proteins to achieve its goals. This study forms the basis for many future investigations on the molecular mitochondrial biology of octocorals as well as other non-bilaterians, augmenting the understanding of the evolution of animal mitochondria, and also its role in cellular and organismal homeostasis in the context of environmental change.

Background Fingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized. Methods Human primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing. Results FTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3). Conclusions We identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.

Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

Specific differences in signaling and antiviral properties between the different Lambda-interferons, a novel group of interferons composed of IL-28A, IL-28B and IL-29, are currently unknown. This is the first study comparatively investigating the transcriptome and the antiviral properties of the Lambda-interferons IL-28A and IL-29. Expression studies were performed by microarray analysis, quantitative PCR (qPCR), reporter gene assays and immunoluminometric assays. Signaling was analyzed by Western blot. HCV replication was measured in Huh-7 cells expressing subgenomic HCV replicon. All hepatic cell lines investigated as well as primary hepatocytes expressed both IFN-λ receptor subunits IL-10R2 and IFN-λR1. Both, IL-28A and IL-29 activated STAT1 signaling. As revealed by microarray analysis, similar genes were induced by both cytokines in Huh-7 cells (IL-28A: 117 genes; IL-29: 111 genes), many of them playing a role in antiviral immunity. However, only IL-28A was able to significantly down-regulate gene expression (n = 272 down-regulated genes). Both cytokines significantly decreased HCV replication in Huh-7 cells. In comparison to liver biopsies of patients with non-viral liver disease, liver biopsies of patients with HCV showed significantly increased mRNA expression of IL-28A and IL-29. Moreover, IL-28A serum protein levels were elevated in HCV patients. In a murine model of viral hepatitis, IL-28 expression was significantly increased. IL-28A and IL-29 are up-regulated in HCV patients and are similarly effective in inducing antiviral genes and inhibiting HCV replication. In contrast to IL-29, IL-28A is a potent gene repressor. Both IFN-λs may have therapeutic potential in the treatment of chronic HCV.

Chronic allograft dysfunction (CAD) following kidney transplantation is characterized by progressive fibrosis and a smoldering inflammatory infiltrate. A modified Fischer 344 (RT1lvl) to Lewis (RT1l) rat renal allograft model was used to study transcriptomic changes during the initiation and progression of CAD and to identify potential therapeutic modes of action of treatment with 13cRA previously shown to limit the development of CAD. Transcriptomic profiling was performed using Affymetrix DNA arrays at time points 0, 7, 14 and 56 days after transplantation. The animal model showed development of significant chronic fibrotic damage with accompanying inflammatory infiltrate by day 56 after transplantation. Regulatory pathways were identified by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and modulated, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database. Microarray analysis revealed dramatic changes in the mRNA expression levels of genes associated with inflammation and fibrosis, as well as the hedgehog and WNT pathways, with a gradual increase in the number of differentially regulated genes during progression of tissue damage. Disease phenotype, as well as differential regulation of select components of the hedgehog, canonical WNT and WNT-Ca2+ signaling pathways could be verified by quantitative PCR (qPCR) and immunohistochemistry. Treatment with 13cRA, not only attenuated disease progression, but even reversed early effects of CAD. The overall effects of the treatment are mediated by a potentially direct influence on fibrosis and inflammation associated gene expression, as well as by a specific modulation, observed for hedgehog and WNT pathway activations. The results identify a series of potential pathways that may represent therapeutic targets in chronic allograft dysfunction.

Background: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. Methods: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema ( E) and 15 controls ( C) undergoing surgery for lung tumor resection or volume reduction ( n = 2). Fibroblasts (8E/9C) were stained for senescence- associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/ 3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. Results: The median ( quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2; 4.7) % in controls and 16.0 (10.0; 24.8) % in emphysema ( p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array ( factor = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein ( IGFBP)-3 and IGFBP-rP1 ( p = 0.029, p = 0.0002), while expression of IGFBP-5, - rP2 ( CTGF), - rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 ( p = 0.006) in emphysema. Conclusion: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence- associated IGFBP-3 and - rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.