Quantitative PCR (qPCR) is the method of choice for accurate estimation of gene expression. Part of its appeal for researchers comes from having a protocol that is easy to execute. However when your reactions do not result in ideal amplification, troubleshooting "why" can be challenging. Factors including sample quality, template quantity, master mix differences, assay design, and incorrect primer or probe resuspension can all influence efficient amplification. When troubleshooting, analysis of the appearance of your amplification curve can give you clues towards improving your results.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
PacBio SMRT - THIRD GENERATION SEQUENCING TECHNIQUEMuunda Mudenda
Nucleic acids sequencing is a very powerful molecular biology and biotechnology technique that gives way to discovery, invention, and solutions. This academic document discusses Single-Molecule Real-Time (SMRT) sequencing platform by Pacific Biosciences (PacBio). The doeucment does not claim to exhaust the subject but you will surely get all the needed highlights to understand this technology better. If you would like an in-depth discussion, do not hesitate to write me an email. Enjoy the read.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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.
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+ )
This presentation covers the introduction to Insect Cell Culture. Also covers its general information about cell culture practices followed in the lab. It covers culture media, the source of cells for culture and examples of the cell line with their culture conditions.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
PacBio SMRT - THIRD GENERATION SEQUENCING TECHNIQUEMuunda Mudenda
Nucleic acids sequencing is a very powerful molecular biology and biotechnology technique that gives way to discovery, invention, and solutions. This academic document discusses Single-Molecule Real-Time (SMRT) sequencing platform by Pacific Biosciences (PacBio). The doeucment does not claim to exhaust the subject but you will surely get all the needed highlights to understand this technology better. If you would like an in-depth discussion, do not hesitate to write me an email. Enjoy the read.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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.
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+ )
This presentation covers the introduction to Insect Cell Culture. Also covers its general information about cell culture practices followed in the lab. It covers culture media, the source of cells for culture and examples of the cell line with their culture conditions.
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.
A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting
Similar to Troubleshooting qPCR: What are my amplification curves telling me? (20)
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.
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
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system.
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.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
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
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
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.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
263778731218 Abortion Clinic /Pills In Harare ,sisternakatoto
263778731218 Abortion Clinic /Pills In Harare ,ABORTION WOMEN’S CLINIC +27730423979 IN women clinic we believe that every woman should be able to make choices in her pregnancy. Our job is to provide compassionate care, safety,affordable and confidential services. That’s why we have won the trust from all generations of women all over the world. we use non surgical method(Abortion pills) to terminate…Dr.LISA +27730423979women Clinic is committed to providing the highest quality of obstetrical and gynecological care to women of all ages. Our dedicated staff aim to treat each patient and her health concerns with compassion and respect.Our dedicated group ABORTION WOMEN’S CLINIC +27730423979 IN women clinic we believe that every woman should be able to make choices in her pregnancy. Our job is to provide compassionate care, safety,affordable and confidential services. That’s why we have won the trust from all generations of women all over the world. we use non surgical method(Abortion pills) to terminate…Dr.LISA +27730423979women Clinic is committed to providing the highest quality of obstetrical and gynecological care to women of all ages. Our dedicated staff aim to treat each patient and her health concerns with compassion and respect.Our dedicated group of receptionists, nurses, and physicians have worked together as a teamof receptionists, nurses, and physicians have worked together as a team wwww.lisywomensclinic.co.za/
Troubleshooting qPCR: What are my amplification curves telling me?
1. Troubleshooting qPCR:
What are my amplification curves telling me?
Aurita Menezes, Ph.D., Scientific Applications Specialist
Integrated DNA Technologies
2. Overview
Basics of an Amplification Curve
Phases of an amplification curve
Terminology
Setting the correct baseline and threshold
Problematic qPCR Curves
No amplification
Unexpected efficiency
Delayed and early Cq
Scattered replicates
Unusual curves
Noisy signal
Amplification beyond plateau
Negative curves
Aurita Menezes
Integrated DNA Technologies
3. Basics of an Amplification Curve
Background
Aurita Menezes
Integrated DNA Technologies
4. R, ΔR, Rn, and ΔRn
R= Multicomponent view
(fluorescence obtained Baseline any normalization)
∆R= Fluorescence - without
Rn: ΔRn = Rn – baseline fluorescence
Normalized reporter signal=emission of the reporter dye
emission of the passive reference dye (ROX)
Aurita Menezes
Integrated DNA Technologies
5. Baseline and Threshold
Linear View Log View
Baseline stop value should be set 1 to 2 cycles before earliest amplification
Set Baseline in Linear View
Set Threshold in Log View
Aurita Menezes
Integrated DNA Technologies
6. Improper Baseline and Threshold
Linear Rn View Log Baselined ΔRn
Aurita Menezes
Integrated DNA Technologies
8. NoNo Amplification
amplification
Incorrectly assigned dye detector
Make sure instrument setting for dye FAM incorrectly assigned as TAMRA
matches dye used in probe
Missing a master mix component
Repeat the experiment
Sample degradation
Does a different cDNA prep give
you the same result?
Lack of target in sample
FAM incorrectly assigned as TET
Test a positive control
Assay design
Try a different assay
Machine not calibrated for dye
Calibrate the instrument
Aurita Menezes
Integrated DNA Technologies
9. Unexpected PCR Efficiency
Lower efficiency (<85%)
Incorrect dilutions causing errors in standard curve
Not enough dynamic range of standard curve
Primers designed on a SNP site
Lower fluorescence of dye
Instrument not calibrated for dye
Sample inhibition
Higher efficiency (>110%)
Incorrect dilutions causing errors in standard curve
Not enough dynamic range of standard curve
Genomic DNA contamination
Incomplete DNase treatment
Aurita Menezes
Integrated DNA Technologies
10. PCR Efficiency
Efficiency reflects whether DNA doubled
every cycle
It takes 3.32 cycles for DNA to be amplified
10 fold
If samples have been correctly diluted, every
10-fold dilution should be 3.32 cycles
Aurita Menezes
Integrated DNA Technologies
13. Delayed Cq……..Lower efficiency
If 10-fold dilutions are all >3.32 cycles apart:
Are your primers on a SNP site?
Consider using IDT PrimeTime® Predesigned Assays designed to avoid SNP
sites through the use of updated sequence information from NCBI databases
Aurita Menezes
Integrated DNA Technologies
14. Delayed Cq……Lower fluorescent dye intensity combined with suboptimal
Efficiency issues
instrument optics for Dye B
Dye A
Dye B
Aurita Menezes
Integrated DNA Technologies
15. Delayed Cq……Sample inhibition
The concentration of inhibitors is maximum in the least dilute
sample
As the sample is diluted, the inhibitory effect decreases
Make a new cDNA prep, try to minimize contamination with phenol layer
during RNA isolation
10-fold dilution
Aurita Menezes
Integrated DNA Technologies
16. Delayed Cq……Master mixes can make a difference
Master Mix A
Master Mix A
Master Mix B Master Mix B
10-fold dilutions
HPRT TBP
Aurita Menezes
Integrated DNA Technologies
17. Early Cq…..Too much template
Too much template
Cq value comes up before cycle 15
True amplification is observed when analyzed in the linear view
Aurita Menezes
Integrated DNA Technologies
18. Early Cq…..Automatic baseline failure
When too much template is present, it’s likely that the instrument’s
software is unable to distinguish between noise and true amplification.
In such cases, auto baseline may assign an incorrect value for the
baseline correction factor.
Adjust the baseline manually to correct this problem
Aurita Menezes
Integrated DNA Technologies
19. Scattered Replicates
Pipetting errors
Poor thermal calibration (thermocycler is raising and lowering
temperature inconsistently across different wells)
Denaturation time is too short (if using a fast cycling master mix,
consider increasing denaturation time from 5 to 20 sec.)
Low copy number
Incorrectly set baseline
Replicates ideally should not be
more than 0.5 Cq apart
Aurita Menezes
Integrated DNA Technologies
20. Height of Amplification Curve
Lowered background
Probe concentration
Signal bleed over
Incorrectly assigned detector
Increased ROX in samples
Master mix
Aurita Menezes
Integrated DNA Technologies
21. Height of Amplification Curve…..
Lowered background due to improved quenching
IDT double-quenched ZEN™ probes (available with IDT PrimeTime®
qPCR Assays) have lower background and increased sensitivity
Aurita Menezes
Integrated DNA Technologies
22. Height of Amplification Curve……Incorrect probe concentration
Correct Probe
Concentration
Incorrect Probe
Concentration
Aurita Menezes
Integrated DNA Technologies
23. Height of Amplification Curve…. Amount of ROX
50 nM ROX
50 nM ROX
100 nM ROX
Noisy signal
10 nM ROX
Aurita Menezes
Integrated DNA Technologies
24. Height of Amplification Curve……Multiplex vs. Singleplex
The height of amplification curve is typically lowered when a target is
investigated in a multiplex reaction vs. a singleplex reaction.
More importantly, it is critical that the Cq is not shifted between both
reactions.
If multiplexing,
The master mix needs to be
adjusted for additional
dNTPs, Mg, and Taq enzyme Singleplex
or Multiplex
Use a master mix
specifically designed for
multiplexing
Aurita Menezes
Integrated DNA Technologies
28. Unusual curve……..Negative curves
If the instrument is not correctly calibrated,
Fluorescence due to amplification increases in a given channel, however the fluorescence
attributed to background will also increase, while fluorescence attributed to the other
dyes and the normalizer may be artificially lowered resulting in negative curves
Calibrate the machine
again for all the dyes
being used
Aurita Menezes
Integrated DNA Technologies
30. Unusual curves….
Amplification is observed beyond plateau
Fluorescence detected is at maximum capacity for the detector
Consequently, the amount of fluorescence attributed to Rox is
mistakebly decreased as the amount of fluorescence attributed to
When ROX normalization is
back ground increases.
turned off,
Consequentlycurve looks normal normalized to a smaller Rox value,
the fluorescence is
artificially increasing the heinght of the amp curve
Turn normalizer off
Aurita Menezes
Integrated DNA Technologies
31. Summary
Information on PrimeTime® qPCR Assays with ZEN™ double-quenched probes and
PrimeTime® qPCR Primers can be found at:
http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr
For background on setting up qPCR experiments, qPCR protocols, and
troubleshooting information like that presented in this webinar, download the IDT
PrimeTime® qPCR Application Guide at:
http://www.idtdna.com/pages/support/technical-vault/reading-room/user-guides-
protocols
Information on products that can be used as controls such as MiniGenes™,
gBlocks™, Ultramers™ and can be found at:
http://www.idtdna.com/pages/products/genes/custom-gene-synthesis
http://www.idtdna.com/pages/products/genes/gblocks-gene-fragments
http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos
Aurita Menezes
Integrated DNA Technologies
34. Threshold
Linear Scale
Logarithmic Scale
Bad Threshold – Good Threshold – Bad Threshold –
in plateau phase in exponential phase in baseline phase
Aurita Menezes
Integrated DNA Technologies
36. Height of Amplification Curve….. Level of ROX
Least ROX ROX Normalization =OFF
High Rox
ROX Normalization=ON
Aurita Menezes
Integrated DNA Technologies
39. Delayed Cq…..High ROX in reaction
Differences in ROX concentration
Aurita Menezes
Integrated DNA Technologies
Editor's Notes
Thank you Hance. AsHancementioned,in my role as the Scientific Application specialist I am expected to support researchers in different aspects of troubleshooting. Often it is just the amplification curve that is provided and I then need to deduce what could possibly have gone wrong with an experiment. So this webinar is aimed to give you some troubleshooting clues as to what can possibly be the issue based on the shape of the amplification curve
Before I discuss various problematic qPCR curves, I am first going to cover the basic phases of an amplification cuve, and give an explanation of some of the commonly used terminology such as R, Delta R etc. We will also discuss the importance of setting the correct baseline and threshold . Finally we will cover troubleshooting various qPCR amplification curves
The 3 main phases of an amplification curve are described in this picture. The baseline region is the time that amplification is occurring , however the amount of fluorescence does not rise above the level of background due to the limitations in sensitivity of the detector and lack of significant accumulation of amplicon In the second phase, fluoresence is observed consistent with exponential amplification.Although not depicted in this picture the next phase is the linear phase wherein reagents are being utilized and amplification is no longer exponential but does continue in a linear fashion before it plateaus.
There is a lot of different ways one can view amp curves.Commonly the X axis is defiined by the cycle number.The Y axis can have the fluorescence data expressed in different waysR is the raw fluorescence data obtained from all the channels that were selected on an instrument. So typically if one has a Fam probe with a Roxmastermix, one would expect to see the fluorecence due to Fam increase with amplification and Rox fluorescence to be a detectable signal but a straight line. If no other dye was used such as in a multiplex, all other channels should show no fluorescnece detected. Thie R view is most useful in troubleshooting issues such as calibration as it tells you the amount of fluoreecence observed by the instrument without any number cruchingDelta R is the same data , but baseline s removed , Rn is the view wherein the raw fluorecence is expressed as a ratio that ha s been normalized to a reference dye such as Rox, so here the background is not removed , Finally delta Rn is where the baseline has been removed ( notice the y axis is 0) as well as the fluorescence is normalized
Setting the correct baseline is important as it determines how much fluoresnece will be subtracted by the software in determining delta Rn.On the left we have the same amp curve in the linear view and on the right hand side, it is set in the log view. Baseline should be set in the linear view and 1 to 2 cycles before amp take s off as you don’t want to subtract more signal than needed.Most softwares automatically set threshold, but typically it is easier to set it in the log view as the exponential phase is best visualized
Here we have an example of an improper baseline as well as threshold. As you can see if baseline is set after amplification is observed in the linear view, the curve is affected as it eliminates part of the amp curve when observed in the log view
The most common qPCR issue is the lack of amplification for which there can be many reasons as listed . If no amplification is observed, I would first check if no mistakes were made in selecting the right detector. Here we have two examples, on the top we have the FAM incorrectly assigned as TAMRA and the bottom it has been incorrectly assigned as TET.Secondly I would repeat the experiment to make sure that one did not forget to add a component during set up as well as try and repeat the experiment with a different template or CDNA prep. Finally it is quite likely that the target is simply not expressed in your sample , so a positive control is absolutely essential , as if one does have a positive control such as a plasmid gene, it helps determine if the problem was the assay or the sample
Another frequent issue is that the pcr efficiency is not as expected. Some of the reasons are listed here, however the most frequent issue that can contribute to both high and low efficiency is errors in dilutions and not having a dynamic range in the generation of a standard curve
During PCR if DNA doubles every cycle then, it Is considered to be 100% efficient. So ideally what this means is that for a DNA template to get amplified 10 fold, it takes 3.32 cycles. So if you are making 10 fold dilutions in the generation of the standard curve, one would expect that the amplification curves would be 3.32 cycles apartAn ideal standard curve will also have all its replicates within 0.5 Cq of each other and i your R2 value reflects how your dilutions and replicates fit on your standard curve. Ideally you should get standard curves with R2 value of 0.99
In the next few slides we are going to discuss these reason for a delayed Cq
Here is an example of a standard curve wherein all the 10 fold dilutions are greater than 3.32 cycles apart. This equally distributed delayed Cq could be due to suboptimal primer design.
In this example the first dilution appears to be more deviant from the rest of the samples on the standard curve