Proposed kinetic improvements to a Zika Biosensor developed by the Collins Lab at MIT that would reduce time of detection by 80% with a cost increase of only $0.01 per reaction.
HIV is a retrovirus that causes AIDS by infecting CD4+ T cells. It is estimated that 33.3 million people were living with HIV at the end of 2009 according to UNAIDS. HIV belongs to the lentivirus genus and has an RNA genome that is reverse transcribed into DNA. Its replication cycle involves attachment to CD4+ cells, uncoating, reverse transcription, integration, gene expression through transcription and translation, assembly of new virus particles, and release of particles from the cell. Accessory proteins such as Vpu, Nef, Vpr, and Vif assist in various stages of the replication cycle and modulation of host immune response.
RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
This document discusses waterborne viruses and their detection in water samples. It provides information on different types of viruses found in aquatic systems, including their size and morphology. Smaller viruses are more difficult to remove from water through physical and chemical treatment. The document then discusses the process of collecting and analyzing water samples to detect viruses, including concentrating the viruses, lysing the cells, amplifying the nucleic acids through PCR, and obtaining quantitative results in real-time. Key aspects of primer and probe design for PCR-based detection are also outlined.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
HIV is a retrovirus that causes AIDS by infecting CD4+ T cells. It is estimated that 33.3 million people were living with HIV at the end of 2009 according to UNAIDS. HIV belongs to the lentivirus genus and has an RNA genome that is reverse transcribed into DNA. Its replication cycle involves attachment to CD4+ cells, uncoating, reverse transcription, integration, gene expression through transcription and translation, assembly of new virus particles, and release of particles from the cell. Accessory proteins such as Vpu, Nef, Vpr, and Vif assist in various stages of the replication cycle and modulation of host immune response.
RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
This document discusses waterborne viruses and their detection in water samples. It provides information on different types of viruses found in aquatic systems, including their size and morphology. Smaller viruses are more difficult to remove from water through physical and chemical treatment. The document then discusses the process of collecting and analyzing water samples to detect viruses, including concentrating the viruses, lysing the cells, amplifying the nucleic acids through PCR, and obtaining quantitative results in real-time. Key aspects of primer and probe design for PCR-based detection are also outlined.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
Application Note: A Simple One-Step Library Prep Method To Enable AmpliSeq Pa...QIAGEN
Targeted amplicon sequencing is a cost-effective, convenient and rapid method for variant detection. This application note outlines a straightforward workflow that uses the QIAseq 1-Step Amplicon Library kit to verify AmpliSeq targeted sequencing assays on the Illumina sequencing instruments. By combining end-repair and ligation, the QIAseq 1-Step Amplicon Library Kit offers a fast and efficient 30-minute procedure for the preparation of high-quality, artifact-free Illumina libraries from any PCR amplicons, including AmpliSeq Panels.
New Progress in Pyrosequencing for Automated Quantitative Analysis of Bi- or ...QIAGEN
Pyrosequencing is a highly flexible technology based on sequencing-by-synthesis for the rapid and quantitative analysis of any type of sequence variation. The real-time output delivers high-resolution sequence information, making pyrosequencing highly suitable for applications ranging from biallelic or multiallelic SNP analysis, DNA methylation quantification to complex mutation analysis of multiple sequence variations in a single run.
In this slideeck, we introduce the new PyroMark Q48 Autoprep system which enables fully automated template preparation integrated in the pyrosequencing workflow. In addition, a new Multiple Primer Dispensation (MPD) strategy is presented which allows fully automated dispensation of sequencing primer, offering a seamless workflow from samples to quantitative genotyping results.
This slidedeck focuses on the following topics
• Pyrosequencing technology and workflow in genotyping analysis
• Introduction into the new PyroMark Q48 Autoprep
• MPD strategy for a seamless automated pyrosequencing workflow
Join us and learn how you can apply the new pyrosequencing system and protocol to your variant analysis or genotyping research
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
Fast and Efficient Post-Bisulfite-Seq Library Construction with QIAseq Ultral...QIAGEN
The document describes a new method called post-bisulfite next-generation sequencing library construction (PBLC) for whole genome bisulfite sequencing (WGBS) that overcomes challenges with traditional WGBS workflows. PBLC involves bisulfite treatment before library preparation, which fragments the DNA so no additional fragmentation is needed. This reduces workflow time and enables epigenomic studies from lower DNA inputs. Comparison studies show PBLC produces higher library yields than traditional WGBS and comparable sequencing results, making it a more efficient and sensitive method for WGBS.
The transcriptome of a cell is not fixed, but is dynamic, and reflects the function or type of the cell, the cell stage or the cell's response to intrinsic and extrinsic influences, such as signaling or stress factors. Only on a single cell level, can you eliminate the biological noise that is inherent to standard gene expression analysis – providing you the insights needed for a deeper understanding of transcription dynamics. In this presentation we delve into the different steps of RNA seq starting from a single cell.
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
Advenced molecular techniques in molecular medical genetics laboratoryPeyman Ghoraishizadeh
The document discusses various molecular techniques used in genetic testing and molecular medical genetics laboratories, including:
1. DNA and RNA extraction from samples like blood and tissue.
2. Polymerase chain reaction (PCR) and its uses like detecting genetic mutations. Real-time PCR allows for quantitative analysis.
3. Techniques like ARMS-PCR, RFLP, and MLPA that can detect mutations, deletions, duplications to diagnose genetic disorders.
The techniques discussed have applications in prenatal diagnosis, forensic analysis like DNA fingerprinting, and identifying genetic causes of diseases. Molecular genetics labs aim to precisely analyze genetic information at the DNA and RNA level.
This document discusses various nucleic acid amplification tests (NAATs) for detecting Chlamydia trachomatis, including PCR, real-time PCR, transcription-mediated amplification (TMA), and strand displacement amplification (SDA). It provides details on commercially available tests like Cobas Amplicor, Roche cobas, Abbott RealTime, Gen-Probe Aptima Combo 2, and BD ProbeTec systems. The advantages and limitations of each method are outlined. Sensitivity and specificity rates for different sample types are generally high, ranging from 92-100% sensitivity and 95-100% specificity.
This document provides an overview of reverse transcription polymerase chain reaction (RT-PCR), including its objectives, introduction, history, principle, protocol for one-step and two-step RT-PCR, technical issues, and literature applications. RT-PCR is a technique that allows detection and quantification of mRNA by first converting RNA to cDNA using reverse transcriptase, then exponentially amplifying the cDNA using PCR. It is often used to detect gene expression and distinguish between infectious and non-infectious viruses or variants in samples. Care must be taken to prevent contamination during sample preparation and RT-PCR.
Analyzing Fusion Genes Using Next-Generation SequencingQIAGEN
Fusion genes are hybrid genes formed by the fusion of two separate genes. Translocation, interstitial deletion and chromosomal inversions are some of the genetic events that can lead to the formation of fusion genes. The occurrence of fusion genes and its implications in cancer have already been known, but the emergence of NGS technology – especially RNA sequencing – offers the potential to detect novel gene fusions. You can learn more about fusion genes and applying NGS to detect them at our upcoming webinar, presented by Raed Samara, Ph.D., QIAGEN’s Global Product Manager for NGS technologies.
In this webinar, Dr. Raed Samara will cover:
1. Fusion genes: what they are and a historical perspective
2. Fusion gene detection: the current status
3. RNA sequencing vs. digital RNA sequencing
4. How to detect and accurately quantify novel fusion genes in your sample
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
Overcome the challenges of Nucleic acid isolation from PCR inhibitor-rich mic...QIAGEN
This presentation will focus on nucleic acid extraction tools developed by QIAGEN that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.
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.
This document discusses molecular diagnostic techniques used in pathology. It describes common techniques like PCR, blotting methods including Southern blot, Northern blot and Western blot, and hybridization techniques such as in situ hybridization and FISH. These techniques allow manipulation and analysis of DNA, RNA and proteins and have applications in neoplastic disorders, infectious diseases, inherited conditions and identity determination. The document provides details on the principles, requirements and procedures of various molecular diagnostic techniques and their uses in hematological and non-hematological malignancies, infectious diseases, inherited genetic disorders and identity determination.
This document summarizes QIAGEN's products for sample preparation, targeted sequencing, and single-cell analysis across various areas of biomedical research including liquid biopsy, circulating tumor cells, and gene expression profiling. Key products mentioned are the QIAseq cfDNA All-in-One Kits for streamlined library preparation from plasma/serum, QIAseq Targeted DNA/RNA Panels for digital sequencing of genomic regions or genes, and QIAseq FX Single Cell DNA/RNA Library Kits for cell-to-library workflows from isolated single cells.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
Application Note: A Simple One-Step Library Prep Method To Enable AmpliSeq Pa...QIAGEN
Targeted amplicon sequencing is a cost-effective, convenient and rapid method for variant detection. This application note outlines a straightforward workflow that uses the QIAseq 1-Step Amplicon Library kit to verify AmpliSeq targeted sequencing assays on the Illumina sequencing instruments. By combining end-repair and ligation, the QIAseq 1-Step Amplicon Library Kit offers a fast and efficient 30-minute procedure for the preparation of high-quality, artifact-free Illumina libraries from any PCR amplicons, including AmpliSeq Panels.
New Progress in Pyrosequencing for Automated Quantitative Analysis of Bi- or ...QIAGEN
Pyrosequencing is a highly flexible technology based on sequencing-by-synthesis for the rapid and quantitative analysis of any type of sequence variation. The real-time output delivers high-resolution sequence information, making pyrosequencing highly suitable for applications ranging from biallelic or multiallelic SNP analysis, DNA methylation quantification to complex mutation analysis of multiple sequence variations in a single run.
In this slideeck, we introduce the new PyroMark Q48 Autoprep system which enables fully automated template preparation integrated in the pyrosequencing workflow. In addition, a new Multiple Primer Dispensation (MPD) strategy is presented which allows fully automated dispensation of sequencing primer, offering a seamless workflow from samples to quantitative genotyping results.
This slidedeck focuses on the following topics
• Pyrosequencing technology and workflow in genotyping analysis
• Introduction into the new PyroMark Q48 Autoprep
• MPD strategy for a seamless automated pyrosequencing workflow
Join us and learn how you can apply the new pyrosequencing system and protocol to your variant analysis or genotyping research
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
Fast and Efficient Post-Bisulfite-Seq Library Construction with QIAseq Ultral...QIAGEN
The document describes a new method called post-bisulfite next-generation sequencing library construction (PBLC) for whole genome bisulfite sequencing (WGBS) that overcomes challenges with traditional WGBS workflows. PBLC involves bisulfite treatment before library preparation, which fragments the DNA so no additional fragmentation is needed. This reduces workflow time and enables epigenomic studies from lower DNA inputs. Comparison studies show PBLC produces higher library yields than traditional WGBS and comparable sequencing results, making it a more efficient and sensitive method for WGBS.
The transcriptome of a cell is not fixed, but is dynamic, and reflects the function or type of the cell, the cell stage or the cell's response to intrinsic and extrinsic influences, such as signaling or stress factors. Only on a single cell level, can you eliminate the biological noise that is inherent to standard gene expression analysis – providing you the insights needed for a deeper understanding of transcription dynamics. In this presentation we delve into the different steps of RNA seq starting from a single cell.
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
Advenced molecular techniques in molecular medical genetics laboratoryPeyman Ghoraishizadeh
The document discusses various molecular techniques used in genetic testing and molecular medical genetics laboratories, including:
1. DNA and RNA extraction from samples like blood and tissue.
2. Polymerase chain reaction (PCR) and its uses like detecting genetic mutations. Real-time PCR allows for quantitative analysis.
3. Techniques like ARMS-PCR, RFLP, and MLPA that can detect mutations, deletions, duplications to diagnose genetic disorders.
The techniques discussed have applications in prenatal diagnosis, forensic analysis like DNA fingerprinting, and identifying genetic causes of diseases. Molecular genetics labs aim to precisely analyze genetic information at the DNA and RNA level.
This document discusses various nucleic acid amplification tests (NAATs) for detecting Chlamydia trachomatis, including PCR, real-time PCR, transcription-mediated amplification (TMA), and strand displacement amplification (SDA). It provides details on commercially available tests like Cobas Amplicor, Roche cobas, Abbott RealTime, Gen-Probe Aptima Combo 2, and BD ProbeTec systems. The advantages and limitations of each method are outlined. Sensitivity and specificity rates for different sample types are generally high, ranging from 92-100% sensitivity and 95-100% specificity.
This document provides an overview of reverse transcription polymerase chain reaction (RT-PCR), including its objectives, introduction, history, principle, protocol for one-step and two-step RT-PCR, technical issues, and literature applications. RT-PCR is a technique that allows detection and quantification of mRNA by first converting RNA to cDNA using reverse transcriptase, then exponentially amplifying the cDNA using PCR. It is often used to detect gene expression and distinguish between infectious and non-infectious viruses or variants in samples. Care must be taken to prevent contamination during sample preparation and RT-PCR.
Analyzing Fusion Genes Using Next-Generation SequencingQIAGEN
Fusion genes are hybrid genes formed by the fusion of two separate genes. Translocation, interstitial deletion and chromosomal inversions are some of the genetic events that can lead to the formation of fusion genes. The occurrence of fusion genes and its implications in cancer have already been known, but the emergence of NGS technology – especially RNA sequencing – offers the potential to detect novel gene fusions. You can learn more about fusion genes and applying NGS to detect them at our upcoming webinar, presented by Raed Samara, Ph.D., QIAGEN’s Global Product Manager for NGS technologies.
In this webinar, Dr. Raed Samara will cover:
1. Fusion genes: what they are and a historical perspective
2. Fusion gene detection: the current status
3. RNA sequencing vs. digital RNA sequencing
4. How to detect and accurately quantify novel fusion genes in your sample
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
Overcome the challenges of Nucleic acid isolation from PCR inhibitor-rich mic...QIAGEN
This presentation will focus on nucleic acid extraction tools developed by QIAGEN that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.
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.
This document discusses molecular diagnostic techniques used in pathology. It describes common techniques like PCR, blotting methods including Southern blot, Northern blot and Western blot, and hybridization techniques such as in situ hybridization and FISH. These techniques allow manipulation and analysis of DNA, RNA and proteins and have applications in neoplastic disorders, infectious diseases, inherited conditions and identity determination. The document provides details on the principles, requirements and procedures of various molecular diagnostic techniques and their uses in hematological and non-hematological malignancies, infectious diseases, inherited genetic disorders and identity determination.
This document summarizes QIAGEN's products for sample preparation, targeted sequencing, and single-cell analysis across various areas of biomedical research including liquid biopsy, circulating tumor cells, and gene expression profiling. Key products mentioned are the QIAseq cfDNA All-in-One Kits for streamlined library preparation from plasma/serum, QIAseq Targeted DNA/RNA Panels for digital sequencing of genomic regions or genes, and QIAseq FX Single Cell DNA/RNA Library Kits for cell-to-library workflows from isolated single cells.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
Method of detection of food borne pathogen(methods).docxOsama Alam
PCR and RT-PCR are commonly used molecular techniques for detecting foodborne pathogens through amplification of pathogen DNA or RNA. Multiplex PCR (mPCR) allows simultaneous detection of multiple pathogens. Real-time PCR monitors amplification in real-time without gel electrophoresis. Other methods like LAMP, NASBA, and microarrays provide isothermal amplification or detect multiple targets but require different primers or probes. Optical and electrochemical biosensors detect binding through surface plasmon resonance or changes in electrical signals. Mass-based sensors measure added mass through resonant frequency changes of piezoelectric crystals. ELISA is a common immunological technique that sandwiches the target antigen between immobilized and enzyme-conjugated antibodies for colorimetric detection.
2012 Ultrasensitive detection of pathogenic bacteria by CRISPR_Cas12a couplin...Jair Téllez
This document describes the development of an ultrasensitive electrochemical biosensor for detecting pathogenic bacteria using a CRISPR/Cas12a system coupled with a primer exchange reaction (PER). The biosensor utilizes functional DNA aptamers that lock a hairpin structure of the PER, preventing primer extension in the absence of target bacteria. The presence of target bacteria unlocks the hairpin, triggering the PER and extending the primer. This produces single-stranded DNA that activates the ssDNase activity of Cas12a, cleaving a methylene blue-labeled probe on an gold electrode and decreasing the electrochemical signal. The biosensor can quantify Escherichia coli O157:H7 from 10 to 106 CFU/mL
2012 Ultrasensitive detection of pathogenic bacteria by CRISPR_Cas12a couplin...Jair Téllez
This document describes the development of an ultrasensitive electrochemical biosensor for detecting pathogenic bacteria using a CRISPR/Cas12a system coupled with a primer exchange reaction (PER). The biosensor utilizes functional DNA aptamers that lock a hairpin structure of the PER, preventing primer extension in the absence of target bacteria. The presence of target bacteria unlocks the hairpin, triggering the PER and extending the primer. This produces single-stranded DNA that activates the ssDNase activity of Cas12a, cleaving a methylene blue-labeled probe on an gold electrode and decreasing the electrochemical signal. The biosensor can quantify Escherichia coli O157:H7 from 10 to 106 CFU/mL
This document provides the timetable and protocols for a practical course on making and analyzing tRNA synthetases in vivo and using cell-free protein synthesis. Over two weeks, students will perform site-directed mutagenesis to produce mutant aminoacyl-tRNA synthetases, express their proteins in E. coli cells and purify the proteins, and use cell-free synthesis to attempt incorporating a phosphotyrosine analogue into a target protein using their mutant synthetases and suppressor tRNA. The document outlines the experimental steps, including mutagenesis, transformation, plasmid preparation, sequencing, protein expression and purification, cell-free reaction set up, and analysis by SDS-PAGE. Safety procedures are also described to handle
Recombinant antibody mediated delivery of organelle-specific DNA pH sensors a...saheli halder
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Lydia Yeshitla, Research Scholar at the Neurobiology Section of UCSDLydia Yeshitla
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1. German Soto, Katie Notarangelo, Anastasia Neuman
Kinetic Engineering Improvements to the Collins’ Lab ZIKA Biosensor
Although Zika rarely causes death in patients, it can be passed from a pregnant mother to
her fetus and cause microcephaly and other severe fetal brain defects. The full complications of
this virus are still unknown, particularly for fetuses. In addition to spread from pregnant mother
to fetus, Zika can be spread through sexual transmission and blood transfusion (Center for
Disease Control and Prevention, 2017). For these reasons, it is important to have a rapid and
inexpensive diagnosis tool for Zika to prevent outbreak originating from uninformed patients and
particularly to reduce spread of the disease to pregnant women. Zika viral loads have been
reported as high as 202 x 10^6 copies/mL (365 fM) in urine (Gourinat et al., 2015). However,
viral loads in saliva and serum are reportedly even lower, with 3 x 10^6 copies/mL (4.9 fM)
(Barzon et al., 2016) documented in patient saliva and 2.5 x 10^6 copies/mL (4.1 fM) (Zika
Experimental Science Team, 2016) and 7.2 x 10^5 copies/mL (1.2 fM) (Lanciotti et al., 2008) in
primate serum, respectively. Previous sensors displayed specificity for their respective Zika
RNA trigger, they were unable to detect clinically relevant RNA concentration. Consequently,
following the recent outbreak of Zika in the United States, the Collins lab developed a sensor to
detect Zika virus using programmable biomolecular components with a much higher specificity,
detecting Zika at concentrations as low as 3 fM (Pardee et al., 2016). The development method
used for this sensor can be translated to other viral diseases, providing a platform for rapid
response to any emerging outbreak.
In the Collin’s Lab Zika biosensor, the analyte being measured is viral Zika RNA. It is
sensed by a programmable RNA sensor called a toehold switch (Figure 1). When the target Zika
RNA binds a complementary region of RNA in the toehold switch, it frees a ribosome binding
sequence and start codon that promote the translation of the reporter gene, LacZ. LacZ mediates
a color change by converting chlorophenol red-beta-D-galactopyranoside (yellow) to
chlorophenol red (purple). The biosensor relies on a colorimetric output that can be read by the
2. naked eye or with a low-cost, battery-powered companion reader. There is an optional, portable
electronic reader that can be paired with the biosensor that provides quantitative measurements.
The electronic reader is manufactured using open-source code, laser-cut acrylic housing, and
readily-available consumer components, such as lithium-ion batteries. An overview of this
process is shown in Figure 2.
Figure 1. The toehold switch in this Zika-detecting device is
designed to complement the Zika trigger RNA. a) The switch RNA
within the toehold is complementary to the trigger RNA, which
here is Zika viral RNA. b) This shows the trigger RNA binding to
the toehold switch RNA. c) When the trigger RNA binds, a
ribosome binds to the ribosome binding site (RBS), and the
previously-repressed LacZ gene is able to be translated. (Image,
Green et al.)
3. Figure 2. The design of the biosensor begins with the development of the toehold switch
chemistry. The toehold is designed to specifically match the trigger sequence of the Zika
RNA, and the dissociation constant (k_D) of the trigger RNA and complementary toehold is
dependent on the length of the binding sequence, which is typically ~30 nucleotides.
Following sample collection, the Zika RNA is extracted and amplified via NASBA. The
paper-based sensor consists of freeze-dried toehold switches, that initiate a colorimetric
change.
The reaction occurring within this sensor that produces a measurable signal is the
conversion of Chlorophenol red-β-D-galactopyranoside to chlorophenol red, mediated by the
enzyme β-galactosidase. Na+
is used as a cofactor for this reaction. This enzyme is coded by the
reporter gene, LacZ, and is translated upon binding of Zika RNA to the toehold switch. The
balanced reaction for this conversion can be found below. The most important feature of this
reaction is the color change; Chlorophenol red-β-D-galactopyranoside has a yellow color while
chlorophenol red has a purple color, allowing the signal to be seen by the naked eye. The
Michaelis Constant (KM)of this β-galactosidase mediated reaction is about 990 umol/L, as
determined experimentally by Maceiczyk et al.
C25H22Cl2O10S (yellow color) + H2O -> C19H12Cl2O5S (purple color) + C6H12O6
4. The signal amplification process, Nucleic Acid Sequence-Based Amplification
(NASBA), is used to increase the sensitivity of the diagnostic platform. NASBA is an isothermal
RNA amplification technique, operating at 41° C, that starts with reverse transcription of the
target RNA (in our case the viral Zika RNA) that is mediated by a sequence-specific reverse
primer to create an RNA/DNA duplex. NASBA creates new target RNA that can be detected by
the toehold switch sensors by allowing allowing a T7 promoter to bind and initiate transcription
of the complementary strand, generating a double-stranded DNA product that serves as a
template for T7-mediated transcription and creation of copies of the target RNA sequence.
Specificity to the Zika genome was proven by testing the sensors with Dengue viral RNA
(51-59% similarity to Zika genome). No response to Dengue virus was seen. The system was
also proven to account for genetic variation among Zika RNA. The switches were shown to
tolerate up to 11% mismatch in nucleotide sequence, triggered successfully by both African and
Asian virus lineages. The possibility of false positive results is further minimized by
CRISPR-Cas9-mediated selection downstream of the amplification.
The researchers added a nucleic acid sequence-based amplification (NASBA) step in
order to increase the sensitivity of the system to meet this relevant range. With the added
NASBA reactions, the sensors reported detection of levels of Zika viral RNA as low as 3 fM.
Efforts were made to design a low-cost device with low-cost tests (Table 1). The process
takes a total of ~ 3.5 hours, with the bulk of the time going toward the NASBA amplification
(Table 2).
Table 1. Cost Estimates
Cost Estimates
NASBA Product $0.51/uL
Device $.10 - $1/test
5. Electronic Reader (Optional) $250
The cost of the device is derived from the 2mm paper disk, chlorophenol
red-β-D-galactopyranoside (Sigma-Aldrich), NEB solution A and B (NEB, PURExpress)
RNAse inhibitor (Roche, 03335402001; 0.5%), and linear DNA constructs encoding the toehold
sensors (.33 nM).
The NASBA Product cost is derived from human serum (Sigma H4522; 7%), Reaction
Buffer (Life Sciences NECB-24; 33.5%), Nucleotide Mix (Life Sciences NECN-24; 16.5%),
RNAse inhibitor (Roche, 03335402001; 0.5%), NASBA primer (2%), nuclease free water
(2.5%), RNA amplicon (20%), and Enzyme Mix (Life Sciences NEC-1-24; 25%).
Table 2. Time Estimates
Assay Time
Viral RNA Extraction 2 minutes
Amplification by NASBA 3 hours
Reactions and activation of signal 30 minutes
The device itself and RNA amplification process were engineered to accommodate
low-resource environments and transport time, as Zika and other viral infections are able to
spread rapidly in such areas without access to laboratory-grade equipment. The electronic reader
was made portable and powered by a rechargeable lithium ion battery. The shelf life of the
paper-based reactions and NASBA reactants were extended using a freeze-drying technique to
preserve the biomolecules during transport. The researchers also confirmed that the NASBA
process could successfully be made low-resource by substituting boiling for the initial heating
step.
It is known that there are several components within blood that are known to inhibit PCR
(Schrader et al., 2012), and similarly affect nucleic acid based diagnostics, including NABSA.
6. The inventors of the device overcome this challenge by diluting the sample (serum or plasma)
into water, which sufficiently removes the inhibitory effect in the diagnostic scheme. However,
this can be troublesome because the dilution step affects the overall sensitivity of the diagnostic
device. The inventors circumvent the detrimental effect of the dilution by increasing the NASBA
reaction time, shown to sufficiently compensate for the reduced sensitivity. An area of
improvement would be the elimination of this dilution step by using a different sample that
contains the virus at high enough concentrations for detection (i.e. saliva or urine) (Barzon et al.,
2016).
Further design improvements could be made via biomolecular, interfacial, or device
considerations. Sensitivity of the device could be increased by the biomolecular design of a
stronger LacZ promoter, which upon activation would produce more β-galactosidase, leading to
a stronger colorimetric signal from a lower analyte concentration. This could be addressed
thermodynamically by looking at the dissociation constant involved. The kinetics of the
β-galactosidase enzyme may be able to be improved.
Based on the Michaelis-Menten model of enzyme mechanics, the rate of production of
chlorophenol red, the reporter molecule, may be able to be increased by increasing the
concentration of substrate, the chlorophenol red-beta-D-galactopyranoside. Additionally, as
β-galactosidase is able to catalyze reactions of many different substrates, it may be possible to
find different reporting reactants that may have better kinetics, leading to shorter reaction times,
or reactants that may be more cost-effective.
One problem to address is the time scale required for the testing. While the timing of the
standardized NASBA procedure is the largest contributor to the length of the test, the reaction
itself has been reported to take over an hour. To determine whether the regime of this device is
diffusion- or kinetic-limited, we looked at the Damkohler number, Da:
7. Here, the reaction rate was defined as the rate constant multiplied by the concentration of
analyte, and the diffusion rate was calculated as the diffusivity of beta-galactosidase in water
multiplied by the interfacial area of the paper biosensor. Diffusivity was estimated using the
Stokes-Einstein equation, assuming a spherical protein (Edward):
Calculations gave a Damkohler number much less than 1, leading us to focus on the kinetics of
the biosensor reaction.
The reporter reaction is an enzyme-mediated colorimetric reaction, catalyzed by the
β-galactosidase enzyme, and can be modelled by the Michaelis-Menten kinetics:
This model provides a quantifiable relation between the rate of the enzymatic reaction
and adjustable parameters. Here, we hypothesized that increasing the concentration of the
chlorophenol red β-D-galactopyranoside substrate in the biosensor would increase the overall
rate of the reaction, therefore decreasing the amount of time required to get a response in use.
Maceiczyk et al. reports Michaelis-Menten constant values for this β-galactosidase
reaction as V_max = 2.18282 +/- 0.13769 and K_M = 0.90921 +/- 0.11661 (Figure _). From
8. these values and the 1.025 mM (0.6 mg/mL) concentration used by the Collins lab, we estimate
the reaction rate as 1.157 units, about 53% of the theoretical V_max. Using the
Michaelis-Menten design equation, we find that increasing the concentration by an order of
magnitude to 6.0 mg/mL, or 10.25 mM, 90% of the theoretical V_max can be achieved. This
would increase the reaction rate by about 80%, saving up to 48 minutes in testing time in the
device, while still reasonably below the solubility limit of chlorophenol red
beta-D-galactopyranoside in water (20 mg/mL).
Figure _. Plotted above is the relationship between reaction rate and concentration of
chlorophenol red beta-D galactopyranoside, based on results from Maceiczyk et al.
The current cost of the chlorophenol red-beta-D-galactopyranoside is $102.50/100 mg, or
$1.025/mg (Sigma-Aldrich). At the original concentration of .6 mg/ml, this would be a cost of
$.615/ml, or $.000615/ul. At the new suggested concentration of 6mg/ml, this would be a cost of
$6.15/ml, or $.00615/ul. The cell-free reactions containing chlorophenol
red-beta-D-galactopyranoside had a total volume of 1.8 ul. Although not all of the volume is this
substrate, we will conservatively estimate a volume of 2ul substrate used per reaction. This gives
an original cost of $.00123/reaction and a new cost of $.0123/reaction, or an increase of
$.01107/reaction. If we assume the lowest total test cost suggested in the paper, $.10/test, this
would be approximately a 10% increase in overall cost. If we assume the highest total test cost,
9. $1/test, this would only be approximately a 1% increase in total cost. This miniscule increase in
cost saves 48 minutes in testing time, making this a worthy investment.
In conclusion, we have improved the design of this biosensor by increasing the
concentration of the chlorophenol red-beta-D-galactopyranoside on the paper sensor by ten-fold,
from .6mg/ml to 6 mg/ml. This increases the rate of the toehold reaction and allows for visual
detection of the presence of Zika RNA in 12 minutes rather than one hour, an 80% decrease in
time. This time-save could allow hospitals to diagnose more patients in a smaller timeframe, and
ensure sick patients receive treatment faster.
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https://www.cdc.gov/zika/index.html
Edward, John T. “Molecular Volumes and the Stokes-Einstein Equation.” ACS Publications,
McGill University, pubs.acs.org/doi/pdf/10.1021/ed047p261?src=recsys.
Gourinat, A.-C., O’Connor, O., Calvez, E., Goarant, C., and Dupont-Rouzeyrol, M. (2015).
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Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Cell 159, 925–939 (2014).
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Maceiczyk, R. M., Hess, D., Chui, F. W. Y., Stavrakis, S., and deMello A. J. (2017). Differential
detection photothermal spectroscopy: towards ultra-fast and sensitive label-free detection in
picoliter & femtoliter droplets. Lab Chip, 17, 3654-3663. DOI: 10.1039/c7lc00946a
Pardee, K., Green, A. A., Takahashi, M. K., Connor, D. H. O., Gehrke, L., Collins, J. J., …
Lambert, G. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable
Biomolecular Components Resource Rapid , Low-Cost Detection of Zika Virus Using
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https://doi.org/10.1016/j.cell.2016.04.059
Schrader, C., Schielke, A., Ellerbroek, L., and Johne, R. (2012). PCR inhibitors - occurrence,
properties and removal. J. Appl. Microbiol. 113, 1014–1026.
Zika Experimental Science Team. (2016). ZIKV-001: Infection of three rhesus macaques with
French Polynesian Zika virus. https://zika.labkey. com/project/OConnor/ZIKV-001/begin.view.
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