Specializing in the design and troubleshooting of qPCR, mutagenesis, and cloning experiments at IDT, Scientific Applications Specialist, Adam Clore PhD, gave this presentation about the intelligent selection of PCR primers. Topics include identifying transcript variants for your target of interest and selecting primers that amplify only specific transcripts. Adam also discussed the growing SNP database and the potential impact of SNPs on qPCR data, how to locate any SNPs that fall in your target amplicon, and the use of free NCBI and IDT tools to help you avoid them.
Transcriptomics is the study of RNA, single-stranded nucleic acid, which was not separated from the DNA world until the central dogma was formulated by Francis Crick in 1958, i.e., the idea that genetic information is transcribed from DNA to RNA and then translated from RNA into protein.
The first genome to be sequenced was that of Haemophilus influenzae in 1995.
The E. coli genome was completely sequenced in 1997.
Yeast (Saccharomyces cerevisiae) (12.8 x 106 bp) and worm (Caenorhabditis elegans) genomes were the first eukaryotic genomes to be sequenced in 1999.
Genomes of Drosophila melanogaster and Arabidopsis thaliana were sequenced in 2000.
Transcriptomics is the study of RNA, single-stranded nucleic acid, which was not separated from the DNA world until the central dogma was formulated by Francis Crick in 1958, i.e., the idea that genetic information is transcribed from DNA to RNA and then translated from RNA into protein.
The first genome to be sequenced was that of Haemophilus influenzae in 1995.
The E. coli genome was completely sequenced in 1997.
Yeast (Saccharomyces cerevisiae) (12.8 x 106 bp) and worm (Caenorhabditis elegans) genomes were the first eukaryotic genomes to be sequenced in 1999.
Genomes of Drosophila melanogaster and Arabidopsis thaliana were sequenced in 2000.
The study of the complete set of RNAs (transcriptome) encoded by the genome of a specific cell or organism at a specific time or under a specific set of conditions is called Transcriptomics.
Transcriptomics aims:
I. To catalogue all species of transcripts, including mRNAs, noncoding RNAs and small RNAs.
II. To determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications.
III. To quantify the changing expression levels of each transcript during development and under different conditions.
ONCOGENE AND PROTOONCOGENE
P53 GENE AND ITS APPLICATION IN CANCER ETIOLOGY
TUMOUR SUPPRESSOR GENE AND BCA AND BAC GENE AND ITS APPLICATION ON THE APOPTOSIS AND DEATH RECEPTORS
Single cell analysis has exploded recently mainly due to the development of high-throughput technologies such as NGS. Single cell analysis is being pursued by researchers in many areas including developmental science, cancer, biomarker discovery and more. This presentation covers some of the recent applications from developed by QIAGEN customers.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
it will help you to understand how the protein microarrays are made, what are the different types and what all purposes they are used for. its very useful ppt
An introduction to the tools and methods used for the bioinformatics analysis of ChIP-Seq data.
Written and delivered for the "Epigenetics and its applications in clinical research" course at the Karolinska Institute in Stockholm, Sweden.
What is PCR
Basic Requirements
Types of PCR
Asymmetric PCR
Applications of PCR
Advantages of PCR
Limitations of PCR
DNA Template
Primers
Taq polymerase
Deoxynucleoside
triphosphates(dNTPs)
Buffer solution
Divalent cations(eg.Mg2+ )
RNase H2-dependent PCR (rhPCR) is a powerful method for increasing PCR specificity and eliminating primer-dimers by using blocked primers and a thermostable RNase H2 from Pyrococcus abyssi (P. abyssi). Primers will only support extension and replication after the blocked portion is cleaved. Cleavage by the RNase H2 enzyme occurs only when primers are bound to their complementary target sequence, thus providing increased specificity. Also, the thermostability of P. abyssi RNase H2 provides a “hot start” capability to the reaction. In this presentation, Dr Joseph Dobosy (senior research scientist in the molecular genetics research division of IDT) gives a detailed explanation of the rhPCR mechanism, offer tips on how to design assays using this powerful technology, and discuss examples of applications that benefit from rhPCR.
The study of the complete set of RNAs (transcriptome) encoded by the genome of a specific cell or organism at a specific time or under a specific set of conditions is called Transcriptomics.
Transcriptomics aims:
I. To catalogue all species of transcripts, including mRNAs, noncoding RNAs and small RNAs.
II. To determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications.
III. To quantify the changing expression levels of each transcript during development and under different conditions.
ONCOGENE AND PROTOONCOGENE
P53 GENE AND ITS APPLICATION IN CANCER ETIOLOGY
TUMOUR SUPPRESSOR GENE AND BCA AND BAC GENE AND ITS APPLICATION ON THE APOPTOSIS AND DEATH RECEPTORS
Single cell analysis has exploded recently mainly due to the development of high-throughput technologies such as NGS. Single cell analysis is being pursued by researchers in many areas including developmental science, cancer, biomarker discovery and more. This presentation covers some of the recent applications from developed by QIAGEN customers.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
it will help you to understand how the protein microarrays are made, what are the different types and what all purposes they are used for. its very useful ppt
An introduction to the tools and methods used for the bioinformatics analysis of ChIP-Seq data.
Written and delivered for the "Epigenetics and its applications in clinical research" course at the Karolinska Institute in Stockholm, Sweden.
What is PCR
Basic Requirements
Types of PCR
Asymmetric PCR
Applications of PCR
Advantages of PCR
Limitations of PCR
DNA Template
Primers
Taq polymerase
Deoxynucleoside
triphosphates(dNTPs)
Buffer solution
Divalent cations(eg.Mg2+ )
RNase H2-dependent PCR (rhPCR) is a powerful method for increasing PCR specificity and eliminating primer-dimers by using blocked primers and a thermostable RNase H2 from Pyrococcus abyssi (P. abyssi). Primers will only support extension and replication after the blocked portion is cleaved. Cleavage by the RNase H2 enzyme occurs only when primers are bound to their complementary target sequence, thus providing increased specificity. Also, the thermostability of P. abyssi RNase H2 provides a “hot start” capability to the reaction. In this presentation, Dr Joseph Dobosy (senior research scientist in the molecular genetics research division of IDT) gives a detailed explanation of the rhPCR mechanism, offer tips on how to design assays using this powerful technology, and discuss examples of applications that benefit from rhPCR.
Meeting the challenges of miRNA research: miRNA and its Role in Human Disease...QIAGEN
miRNA plays a critical role in many biological processes such as differentiation and development, cell signaling, response to infection and more. This slideshow will cover the biology of miRNA, the key challenges associated with miRNA research and the latest advances in miRNA research technology.
Introduction to 16S rRNA gene multivariate analysisJosh Neufeld
Short introductory talk on multivariate statistics for 16S rRNA gene analysis given at the 2nd Soil Metagenomics conference in Braunschweig Germany, December 2013. A previous talk had discussed quality filtering, chimera detection, and clustering algorithms.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Multiplex Ligation- Dependent Probe Amplification (50 kadar farklı genomik DNA veya RNA dizisindeki normal olmayan kopya sayısının tespitini sağlayan bir multipleks PCR yöntemi)
STR DNA profiling is now a powerful, inexpensive tool that can generate unique DNA signatures that can be used to authenticate cell lines and detect contamination of more than one cell type. This presentation will talk about why scientists need cell authentication, what is STR profile and STR profile workflow from Creative Bioarray.
The human genome is full of repeated DNA sequences which come in various sizes and are classified according to the length of the core repeat units, the number of contiguous repeat units, and/or the overall length of the repeat region. DNA regions with short repeat units (usually 2-6 bp in length) are called Short Tandem Repeats (STR).
Creative Bioarray STR profiling is critical for verifying the identity of human cell lines, ensuring uniqueness of the cell line and detecting laboratory errors such as misidentification and cross-contamination of lines. The sensitivity and high power of discrimination makes our STR analysis an ideal choice for the various types of cell authentication.
https://www.creative-bioarray.com/Services/Short-Tandem-Repeat-Analysis.htm
The human genome is full of repeated DNA sequences which come in various sizes and are classified according to the length of the core repeat units, the number of contiguous repeat units, and/or the overall length of the repeat region. DNA regions with short repeat units (usually 2-6 bp in length) are called Short Tandem Repeats (STR).
Visualization Approaches for Biomedical Omics Data: Putting It All TogetherNils Gehlenborg
Keynote Talk presented at the 1st Annual BiVi Community Annual Meeting (17 December 2014)
http://bivi.co/page/bivi-annual-meeting-16-17th-december-2014
Visualization Approaches for Biomedical Omics Data: Putting It All Together
The rapid proliferation of high quality, low cost genome-wide measurement technologies such as whole-genome and transcriptome sequencing, as well as advances in epigenomics and proteomics, are enabling researchers to perform studies that generate heterogeneous datasets for cohorts of thousands of individuals. A common feature of these studies is that a collection of genome-wide, molecular data types and phenotypic or clinical characterizations are available for each individual. These data can be used to identify the molecular basis of diseases and to characterize and describe the variations that are relevant for improved diagnosis, prognosis and targeted treatment of patients. An example for a study in which this approach has been successfully applied is The Cancer Genome Atlas project (http://cancergenome.nih.gov).
In my talk I will discuss how visualization approaches can be applied to enable exploration and support analysis of data generated by such studies. Specifically, I will review techniques and tools for visual exploration of individual omics data types, their ability to scale to large numbers of individuals or samples, and emerging techniques that integrate multiple omics data types for interactive visual analysis. I will also examine technical and legal challenges that developers of such visualization tools are facing. To conclude my talk, I will outline research opportunities for the biological data visualization community that address major challenges in this domain.
FORENSIC DNA PROFILING: Strengths and LimitationsHezekiah Fatoki
Forensic science is defined as the application of scientific knowledge and experimentation to legal contentions, be they civil or criminal matters. DNA profiling (also called DNA typing or DNA fingerprinting) is a forensic techniques used to identify individuals by characteristics of their DNA in crime cases. DNA profiling can be use to resolve paternal and ancestral issues. This process was built mainly on the knowledge of two scientific breakthroughs. First is the Polymerase Chain Reaction (PCR) which was conceived by Kary Mullis in 1983 at Cetus Corporation, USA. Second is the Restriction Fragment Length Polymorphism (RFLP) analysis of repeated DNA sequences which was discovered by Professor Sir Alec Jeffreys in 1985 at the University of Leicester, UK. The strengths and limitations of the current and emerging forensic DNA profiling are the focus of this seminar. It is my expectation that the newly proposed synthetic human genome project will aid the strength of this process in the future.
Use of CRISPR-Cas9 has revolutionized targeted genome editing. However, rapid design of high-quality guide RNA (gRNA) sequences with high on-target and low off-target editing remains challenging. We implemented a machine learning algorithm to design high-quality gRNA sequences in 5 commonly used species (human, mouse, rat, zebrafish, and nematode). Our tool also designs gRNA sequences against custom targets, and can check existing gRNA designs for quality. In this webinar, we review our data illustrating this tool's performance and demonstrate its use in predicting and designing improved gRNAs for genome editing.
Advances in next generation sequencing enable the detection of variants at exceptionally low frequencies. The accurate detection of low-frequency variants is challenging due in part to errors that are introduced during sample preparation, target enrichment, and sequencing. After tagging individual DNA library molecules with adapters containing unique molecular identifiers (UMIs), bioinformatic filters can be applied to identify and correct errors introduced during the sequencing workflow. In this presentation, we walk through the analytical workflows developed at IDT for processing data containing UMIs. We highlight methods to extract UMI information, correct errors, and build consensus among multiple observations of an original source molecule.
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) from patient plasma is becoming more widespread in oncology clinical trials. The noninvasive nature of acquiring these samples is particularly important when resection of representative tumor samples is not advised or not possible. However, profiling of ctDNA has challenges to overcome, such as low concentration of ctDNA shed from the tumor and a low signal:noise ratio caused by somatic alterations with less than 1% variant allele fraction. Improving the sensitivity of these assays to detect low allele frequency events with high confidence requires robust sequencing of low input libraries while employing error correction to reduce background noise. To overcome these challenges, we have incorporated unique molecular identifiers (UMIs) into our NGS workflow. Using these novel adapters paired with our proprietary bioinformatics pipeline (AstraZeneca), the number of false positive variants reported for allele fractions less than 0.5% was reduced tenfold. We also refined our curation based on the mapping quality and strand bias in the vicinity of each variant to further reduce the background noise. The use of xGen® Dual Index UMI Adapters—Tech Access (Integrated DNA Technologies) has enabled us to sequence thousands of plasma samples from diverse tumor indications and at differing time points during our trials. The generated data are highly informative with the potential to answer critical questions relating to individual response or resistance to experimental therapies. During this webinar, we discuss our current NGS ctDNA workflow and our future plans to increase our sequencing sensitivity with these novel UMI adapters.
Single-nucleotide polymorphisms (SNPs) provide important information about the biology and evolution of different organisms. SNPs may also help predict an individual’s response to certain drugs, susceptibility to environmental factors, and risk of developing particular diseases providing valuable insight into pathophysiology of the human condition. As a result, SNPs with important functional roles often become subjects for high-throughput experiments.
In this webinar, Daniel Tsang provides an overview of genotyping using real-time PCR (qPCR) technology, including challenges and ways to overcome these challenges. He presents a novel qPCR-based genotyping solution, the rhAmp™ SNP Genotyping System, along with its advantages in genotyping, details on cluster separation, as well as solutions to improve the calling accuracy and confidence of making genotype calls.
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
The rapid increase in throughput of next generation sequencing (NGS) platforms is changing the genomics landscape. Typically, adapters containing sample indexes are added during library construction to allow multiple samples to be sequenced in parallel. Some strategies also introduce a unique molecular identifier (UMI) within the adapter to correct for PCR and sequencing errors. When a UMI is added, reads are assigned to each sample based on their associated sample index, and the UMI is used for error correction during data analysis. For simplicity, a single adapter that is suitable for a variety of applications would be ideal.
xGen® Dual Index UMI Adapters take the guesswork out of adapter design and ordering. These adapters, created for Illumina sequencers, are compatible with standard library preparation methods and may be sequenced in different modes depending on your application. In addition to unique, dual indexes, the adapters contain a molecular barcode in an optional read position. We will discuss how unique, dual indexes mitigate sample index hopping for multiplexed sequencing and demonstrate how UMIs reduce false positives to improve detection of low-frequency variants.
Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form: late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variants [1].
To help identify causative genetic variants, we have combined highly accurate, long-read sequencing with hybrid-capture technology. In this collaborative webinar*, we present this method and show how combining IDT xGen® Lockdown® Probes with PacBio SMRT® Sequencing allows targeting and sequencing of candidate genes from genomic DNA and corresponding transcripts from cDNA. Using a panel of target capture probes for 35 AD candidate genes, we demonstrate the power of this approach by looking at data for two individuals with AD. Some additional benefits of this method include the ability to leverage long reads, phase heterozygous variants, and link corresponding transcript isoforms to their respective alleles.
Reference: 1. Van Cauwenberghe C, Van Broeckhoven C, Sleegers K. (2016) The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med, 18(5):421–430.
* This presentation represents a collaboration between Pacific Biosciences and Integrated DNA Technologies. The individual opinions expressed may not reflect shared opinions of Pacific Biosciences and Integrated DNA Technologies.
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
Genomics research and discovery has led to a large increase of reported single nucleotide polymorphisms (SNPs). From 2006 to 2017, the number of refSNPs in the NCBI dbSNP database has increased 13-fold. Many polymorphisms can be linked to disease susceptibility and responses to chemical therapies. Other polymorphisms are used as trait identifiers in livestock and plants. Being able to inexpensively and accurately determine the genotype in high-throughput fashion, with low sample input is a critical need in current, large-scale screening efforts. In this presentation, we present a novel, probe-based, PCR genotyping solution that possesses the universal cycling conditions, strong signal generation, and benchtop reaction stability needed for high-throughput screening. We also present the mechanism and unique technical advantages of using the rhAmp SNP Genotyping System, and we will illustrate how easy it is to generate high quality genotyping data.
The increasing throughput of NGS platforms has fueled the demand to sequence many samples in parallel, also referred to as multiplex sequencing. During multiplex sequencing, the identity of each sample library within a pool is maintained using index sequences that are subsequently separated in a process called demultiplexing during data analysis. Historically, a relatively small number of unique sequences (8 x i5 and 12 x i7) were used to create index combinations to multiplex samples. Unfortunately, with this combinatorial approach, a single index swap may cause a read to be mis-assigned to a different sample causing cross-talk. In this presentation, we discuss some sources of sample cross-talk, including index hopping during cluster amplification or multiplexed capture, and how index sequencing errors may lead to demultiplexing mistakes. We discuss how sample cross-talk causes demultiplexing errors and present a method for increasing the accuracy of sample identification using unique, dual-matched index adapters.
As next generation sequencing has moved into the clinic, there is an increased demand for accuracy and reproducibility. Target enrichment is needed for applications where high read depth is critical, but some performance limitations, especially in GC-rich regions of the genome, have raised questions about the overall usefulness of target capture methods. In this presentation, Dr Kristina Giorda presents a method using individually synthesized and quality checked capture baits that performs well, even for GC-rich sequences, and delivers accurate coverage of the target space. Dr Giorda covers library preparation and target capture, and shares informative data generated using our xGen® Exome Research Panel.
CRISPR has become an increasingly popular tool for genome editing, in part because it is highly flexible and relatively easy to implement compared to other technologies. However, for scientists beginning to work with this method, the wide range of products and variety of editing approaches can be overwhelming. In this presentation, Justin Barr provides a simple explanation of the steps for planning your experiment, including guide RNA design, an overview of delivery methods, and options for measuring editing results. He also discusses how to generate specific mutations in the genome using homology-directed repair (HDR).
The CRISPR-Cpf1 nuclease is the best alternative to the commonly used Cas9 for genome editing. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence of TTTV, which differs from the Cas9 PAM sequence, NGG. Having Cpf1 as a second option increases the likelihood of editing as close as possible to your desired target site. In this presentation, Dr Rolf Turk introduces the optimized Alt-R™ CRISPR-Cpf1 System and explains how it can be used as a powerful new tool for your genome editing research. Dr Turk presents the basics of the system, as well as protocols for getting started in genome editing.
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.
Precision medicine for oncology requires accurate and sensitive molecular characterization. However, sample degradation, polymerase errors, and sequencing errors reduce accuracy for sequencing genetic variants. By incorporating molecular tagged adapters in target enrichment, and using DNA probes that deliver extremely even and deep coverage, we are able to demonstrate a 300-fold reduction in false positives at or above 0.25% variant frequency. In this presentation, Dr Mirna Jarosz discusses these methods and how they can significantly reduce error rates in your sequencing data.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
The National Center for Biotechnology Information (NCBI) provides one of the most extensive sets of web-based tools for biological research. The tools are indispensable when planning genomics experiments, including for qPCR, NGS, and CRISPR. In this presentation, Dr Matt McNeill takes a practical look at getting started with the wealth of NCBI tools, and shares some relevant tips to help you sift through the tools and options that we regularly use. In particular, he focuses on commonly adjusted parameters that will allow you to more effectively use the powerful Basic Local Alignment Algorithm Tool (BLAST) to identify off-target hybridization/annealing events. Dr McNeill also covers practical examples using NCBI tools to design assays.
The availability of affordable, high quality, custom gene synthesis has greatly expanded what is possible for labs in numerous research areas. IDT offers a variety of gene synthesis solutions, including our revolutionary double-stranded gBlocks Gene Fragments. In this presentation, Dr Adam Clore discusses the many applications of gBlocks Gene Fragments, such as controls for qPCR and next generation sequencing, and donor templates for homology directed repair in CRISPR experiments. Learn more at www.idtdna.com/gblocks
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
2. Considerations When Designing RT-PCR Assays
Physical Properties of the Primers
and Probe
Transcript Variants
SNPs
Adam Clore, PhD
Finding the Right Primers
3. What is a Transcript Variant and Why Should You Care?
Genes with different
combinations of exons
Alternatively spliced transcripts
produce different isoforms of
proteins
95% of human genes have
alternate splicing
Many cancers and some genetic
diseases are caused by transcript
variants
Adam Clore, PhD
Finding the Right Primers
Gene
Exons
Introns
Differential
Splicing
Transcript
Variants
4. Role of Variants in Normal Gene Expression
Doublesex gene in D. melanogaster influences sex development by
alternatively splicing the 3 exons of the gene:
Pre-mRNA
Female variant
Male variant
Adam Clore, PhD
Finding the Right Primers
5. Role of Transcript Variants in Disease
correlating with cancer:
CD44
Wilms’ tumor gene WT1
BRCA1
MDM2
FGFR
Adam Clore, PhD
Finding the Right Primers
6. NCBI Gene GUI
Use to see all known transcript variants in
the RefSeq database and how they differ.
Adam Clore, PhD
Finding the Right Primers
13. Options for Real Time PCR Design
PrimeTime® Predesigned qPCR Assay Library
For Human, Mouse, and Rat
All designs are screened for crosstalk between variants
http://www.idtdna.com/order/predesignedassay.aspx?source=scitools
RealTime Design Tool
Real-time design for sequences entered by Ref Seq number or sequences
pasted in manually
http://www.idtdna.com/scitools/Applications/RealTimePCR/
Adam Clore, PhD
Finding the Right Primers
14.
15.
16. Exon Naming…
NCBI has had no official convention
An example of how it’s often done
Variant #2
Variant #1
1 2 3 4 5
1 2 3 4 5 6
Adam Clore, PhD
Finding the Right Primers
17. Exon Naming…
What NCBI (and IDT) are moving to:
Variant #2
Variant #1
1 2 3 4 7 8
1 5 6 7 8
Adam Clore, PhD
Finding the Right Primers
18.
19.
20.
21.
22. What is a SNP and Why Do I Care?
Single nucleotide
polymorphism a single letter
change in the genetic code
There are an approximately
56,000,000 SNPs in the human
genome
16,000,000 are in gene introns
and exons
Most are silent mutations
Some are clinically relevant
Adam Clore, PhD
Finding the Right Primers
33. Summary
IDT RT-PCR Design Tools
PrimeTime® Predesign aPCR Assay Tool
For Human, Mouse, or Rat
Identifies transcript variants and avoids SNPs in all Pre-designed Assays
RealTime Design Tool
Can design for any sequences submitted by user
User needs to check for transcript variants and SNPs
Adam Clore, PhD
Finding the Right Primers