This white paper describes SABiosciences' Oligo GEArray DNA microarray system. Key points:
1) Oligo GEArrays profile gene expression of focused gene panels (113-440 genes) related to biological pathways or disease states, providing a more economical alternative to genome-wide microarrays.
2) The system uses flexible target labeling kits, membrane arrays, and a simple chemiluminescent detection method, allowing it to be easily adopted in any laboratory.
3) Data shows the Oligo GEArray has high sensitivity (can detect 10 fM targets), a wide dynamic range, and is highly reproducible, with technical replicates yielding CVs less than 10%.
Gyros Protein Technologies is one of the leading platform for the Nanoliter-Scale Immunoassays.
Specialties in Immunoassays in the biotherapeutic workflow, peptide synthesis for academic and industrial applications.
FirstSource Laboratory Solutions is one of the authorized suppliers of Gyros Immunoassays.
For more details visit us at https://www.firstsourcels.com/gyrolab
Fcγ-RIII immobilized resin was developed for chromatography. Fcγ-RIII binds the Fc portion of IgG proteins and can separate monoclonal antibodies based on glycan structure and ADCC activity. A non-glycosylated, acid-stable version of Fcγ-RIII was produced in E.coli and immobilized on a resin for use in an analytical column to evaluate monoclonal antibody properties and perform quality control comparisons between antibody lots.
The document discusses Cignal Reporter Assays, which are cell-based assays for analyzing gene expression and signaling pathways. The assays use dual-luciferase technology and transcription factor-targeted response elements to provide sensitive and reproducible measurements of 45 signaling pathways. Key advantages of the assays include minimizing experimental variability through dual-luciferase normalization, increasing signal-to-noise ratio using destabilized and codon-optimized luciferase, and maximizing response using optimized transcriptional response elements tailored to each pathway. The assays are available in multiple formats including plasmid, lentiviral, luciferase and GFP, making them suitable for a variety of experimental systems and applications.
Next-generation sequencing technologies have rapidly advanced since 2005. Key developments include massively parallel sequencing reactions that enabled sequencing of entire human genomes for less than $1,000 by 2015. While Illumina dominates the market, other platforms like Ion Torrent and PacBio are increasing capabilities. Routine human whole genome sequencing is now used in research and medicine, enabling new opportunities like liquid biopsies and single-cell analysis. However, data storage and analysis remain challenges due to the large volumes of sequencing data.
The document describes Cignal Reporter Assays from SABiosciences that enable simple and robust analysis of signal transduction pathways. The assays utilize dual-luciferase reporters containing optimized transcriptional regulatory elements and luciferase variants to provide high sensitivity and low variability. The assays allow monitoring of 29 pathways and are available in different formats for various cell types and applications like RNAi and small molecule screening.
The document describes xGen Predesigned Gene Capture Pools, which are pools of xGen Lockdown Probes designed to target specific genes. The pools offer advantages over custom panels such as better customization ability, decreased optimization time, and lower cost. They can be used to build custom gene panels, supplement existing panels, and share targets between researchers studying different diseases. Data shows the pools provide high on-target performance, consistent deep coverage, and can supplement existing panels without affecting coverage.
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
This document discusses QIAGEN's products for microbiome research including sample preparation kits, automation, next-generation sequencing (NGS) library preparation kits, and data analysis software. It highlights two key innovations in their sample prep kits: optimized bead beating for tough sample lysis and inhibitor removal technology to purify nucleic acids. A range of kits are available for isolating DNA and RNA from difficult sample types like soil, water, biofilms, and stool. Library preparation kits make NGS accessible and several kits are compatible with QIAcube for automated sample prep. Data analysis software assists with comparative microbiome profiling and assembly.
Gyros Protein Technologies is one of the leading platform for the Nanoliter-Scale Immunoassays.
Specialties in Immunoassays in the biotherapeutic workflow, peptide synthesis for academic and industrial applications.
FirstSource Laboratory Solutions is one of the authorized suppliers of Gyros Immunoassays.
For more details visit us at https://www.firstsourcels.com/gyrolab
Fcγ-RIII immobilized resin was developed for chromatography. Fcγ-RIII binds the Fc portion of IgG proteins and can separate monoclonal antibodies based on glycan structure and ADCC activity. A non-glycosylated, acid-stable version of Fcγ-RIII was produced in E.coli and immobilized on a resin for use in an analytical column to evaluate monoclonal antibody properties and perform quality control comparisons between antibody lots.
The document discusses Cignal Reporter Assays, which are cell-based assays for analyzing gene expression and signaling pathways. The assays use dual-luciferase technology and transcription factor-targeted response elements to provide sensitive and reproducible measurements of 45 signaling pathways. Key advantages of the assays include minimizing experimental variability through dual-luciferase normalization, increasing signal-to-noise ratio using destabilized and codon-optimized luciferase, and maximizing response using optimized transcriptional response elements tailored to each pathway. The assays are available in multiple formats including plasmid, lentiviral, luciferase and GFP, making them suitable for a variety of experimental systems and applications.
Next-generation sequencing technologies have rapidly advanced since 2005. Key developments include massively parallel sequencing reactions that enabled sequencing of entire human genomes for less than $1,000 by 2015. While Illumina dominates the market, other platforms like Ion Torrent and PacBio are increasing capabilities. Routine human whole genome sequencing is now used in research and medicine, enabling new opportunities like liquid biopsies and single-cell analysis. However, data storage and analysis remain challenges due to the large volumes of sequencing data.
The document describes Cignal Reporter Assays from SABiosciences that enable simple and robust analysis of signal transduction pathways. The assays utilize dual-luciferase reporters containing optimized transcriptional regulatory elements and luciferase variants to provide high sensitivity and low variability. The assays allow monitoring of 29 pathways and are available in different formats for various cell types and applications like RNAi and small molecule screening.
The document describes xGen Predesigned Gene Capture Pools, which are pools of xGen Lockdown Probes designed to target specific genes. The pools offer advantages over custom panels such as better customization ability, decreased optimization time, and lower cost. They can be used to build custom gene panels, supplement existing panels, and share targets between researchers studying different diseases. Data shows the pools provide high on-target performance, consistent deep coverage, and can supplement existing panels without affecting coverage.
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
This document discusses QIAGEN's products for microbiome research including sample preparation kits, automation, next-generation sequencing (NGS) library preparation kits, and data analysis software. It highlights two key innovations in their sample prep kits: optimized bead beating for tough sample lysis and inhibitor removal technology to purify nucleic acids. A range of kits are available for isolating DNA and RNA from difficult sample types like soil, water, biofilms, and stool. Library preparation kits make NGS accessible and several kits are compatible with QIAcube for automated sample prep. Data analysis software assists with comparative microbiome profiling and assembly.
Sequential Automation of RNA and DNA preps on the same QIAcube instrumentQIAGEN
Automation of QIAGEN spin-column kits on the QIAcube saves valuable time and ensures standardized results. Since the same QIAcube may be used by multiple researchers for different applications, cross-contamination between samples and preparation technologies must be avoided (e.g., when nucleases are used). The unique instrument design and features minimize contamination between sequential preps, allowing both RNA and DNA preps to be performed on the same instrument. To show the process safety and robustness, we performed alternating automated RNA preps (requiring a DNase step) and DNA plasmid preps (requiring an RNase step). The preps were sequentially performed on the same QIAcube instrument using the RNeasy® Mini Kit and the QIAprep® Spin Miniprep Kit, respectively.
Independently, we performed a series of manually processed preps to compare with the automated preps. RNA and DNA quality and yields were similar between the two methods, showing the absence of carryover of nucleases.
Accurate quantitation of microRNA is critical for pharmacological and diagnostic studies. A multiplexed bead-based method allows simultaneous generation of standard curves for multiple microRNAs, reducing reagents and labor compared to other methods. The method produced standard curves for 9 microRNAs simultaneously using a single 23-well plate in 4 hours, with coefficients of variation below 5% and the ability to test 74 tissue samples on the same plate.
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.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
The document describes the phage display technique, which involves genetically fusing a protein to the coat protein of a bacteriophage virus. This allows for the display of large protein and peptide libraries on the surface of the phage. The technique has various applications, including epitope determination, enzyme engineering, organ targeting, and isolating allergens. It involves creating a vector fusion, binding and selecting phages to a target, washing, eluting bound phages, and amplifying the process through bacterial infection.
QIAcube® RNA isolation from stool samples using the RNeasy® PowerMicrobiome® ...QIAGEN
This application note demonstrates that RNA is extracted efficiently from stool samples using the RNeasy PowerMicrobiome Kit and the QIAcube system. Furthermore, the RNA isolated with the RNeasy PowerMicrobiome Kit and the QIAcube system is compatible with downstream applications.
NEBNext Ultra DNA for Illumina NGS (ChIP-seq and HLA)_Biomek FXP Automated Wo...Zachary Smith
This document describes the automation of the NEBNext Ultra DNA Library Preparation Kit for Illumina on the Beckman Coulter Biomek FXP liquid handler. The automation allows preparation of up to 96 individually indexed DNA sequencing libraries in approximately 4.5 hours. An intuitive user interface allows specification of sample number and size selection options. Quality control analysis showed the automated libraries had over 1.4 million reads per sample, low contamination, and average insert sizes of 400 bp, demonstrating the method produces high quality libraries.
Assessment of Y chromosome degradation level using the Investigator® Quantipl...QIAGEN
Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.
Ernesto Picardi – Bioinformatica e genomica comparata: nuove strategie sperim...eventi-ITBbari
Bioinformatica e genomica comparata: nuove strategie sperimentali e computazionali per la produzione e analisi di dati NGS finalizzati a sviluppare processi e prodotti innovativi per la salute dell’uomo, l’ambiente e l’agroalimentare.
A Turn-Key Flow-Through-Mode Purification Process to improve Quality and Safe...Merck Life Sciences
In this webinar, you will learn:
Intensified plasma Immunoglobulin purifications
Scalable process development with latest technologies
Improved safety and quality of plasma IgG meeting required quality attributes
Detailed description:
Plasma-derived immunoglobulins (IgG) are essential medicines that are in worldwide shortage. How to develop optimized processing steps for robust and efficient manufacturing has been a constant goal, to make the most out of the precious plasma raw material.
In this study, we present a worse-case equivalent of plasma intermediate, explore various process steps along the fractionation flow, including flow-through-mode chromatography, affinity chromatography, virus inactivation steps and removal of solvent/detergent, single-pass TFF (SPTFF), clarification, and aseptic filtration, to establish a robust, easy-to-operate, readily scalable plasma IgG process with over 99% purity, depletion of IgA, isoagglutinin, and thrombogenic markers, meeting the commonly required 20% concentration for subcutaneous IgG infusion. Such solutions would be appropriate for various IgG intermediates which help to improve the global supply of immunoglobulins.
Next-generation sequencing (NGS) has revolutionized the way we analyze diseases and commercial outfits such as Illumina, Helicos, QIAGEN and Pacific Biosciences have made significant contributions. In addition, the launch of direct-to-consumer genetic testing solutions has dramatically changed the way consumers access genomics data. Until a few years ago, the cost of sequencing was a major bottleneck. Recent developments have reduced the cost from thousands of dollars to a couple of cents per megabase. When did these changes start? What were the changes in the commercial sector in the last 15 years? This infographic is a timeline of the NGS commercial marketplace.
Breaking the Status Quo: Using Mass Spectrometry to detect Host Cell ProteinsMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3b3Tbcd
Measurement of host cell proteins is vital to ensuring a biotherapy's purity and a patient's safety. Biotherapies treat diseases with products produced by living organisms, as a result, host cell components must be characterized and controlled. We'll review new methods within product characterization for detection.
Trace amounts of host cell proteins can be present after the production and purification of any biopharmaceutical. Detection of these species requires highly specific techniques to accurately quantify even low levels of contamination. Host cell protein impurities, present at PPM-levels in biotherapies, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. Their complex and diverse nature makes them challenging to detect or monitor. With acceptance criteria for host residual DNA usually set at a very low level (often =1.0 pg of DNA per mg of drug substance), effective removal techniques and sensitive methods of detection are critical.
Antibody-based techniques, like the enzyme-linked immunosorbent assay (ELISA), have been used to assess the HCP load of biotherapeutics before and after process changes. However, these techniques do not necessarily detect qualitative changes in the HCP population. In this webinar, we will discuss how mass spectrometry (MS)-based approaches coupled with ELISA methods help detect qualitative and quantitative differences in HCP populations.
In this webinar, you will learn:
• Comprehensive HCP ID and semi-quantitation
• HC agnostic process
• Creation of process specific database
• Differential clearance of specific HCPs throughout purification steps
• Monitoring of problematic species e.g. immunogenic (PLBL2), lipases and proteases
• Explanation about why 90% of BLAs filed included this HCP MS data
Digital PCR for soybean GMO detection on the OpenArray Platform: a case study...Thermo Fisher Scientific
This document describes how digital PCR on the TaqMan OpenArray platform can be used as a sensitive tool for detecting genetically modified organisms (GMOs). [The summary describes:]
1) Custom TaqMan assays were designed to differentiate between GMO and wild-type soybean DNA. 2) The assays were validated for specificity using digital PCR. 3) Spike-in experiments were conducted adding GMO DNA at levels as low as 0.01% to wild-type DNA, and digital PCR accurately measured ratios matching estimated levels.
Monoclonal antibody (mAb) therapeutics have formed and continue to form the vast majority of biopharmaceutical company pipelines today with a number of remarkable commercial successes. The advent of mAbs as therapeutics has been greatly aided by a process platform approach that has enabled rapid development and manufacturing for this class of drugs.Downstream process platforms for mAbs first evolved over a decade ago and have had a significant impact on the time and resources spent in process development. This chapter describes some of the platform approaches first used in the biopharmaceutical industry and how those platforms have evolved over the last decade based on needs as well as newly available technology. We also describe the advent of next generation mAb based constructs and the creation of possible platforms for those moieties.
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.
Technology Trends in Bioprocessing PurificationMilliporeSigma
This presentation reviews current trends in bioprocessing purification and includes key considerations for continuous processing and connected polishing for monoclonal antibodies. Topics include:
• Market trends and the evolution of next-generation processes
• Intensified capture processing
• Continuous virus inactivation
• Connected flow-through polishing
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab
Validation of anti niv igm capture elisa version#1krishgen
NiV is a negative-sense, non-segmented RNA virus that was first isolated from cerebrospinal fluid of human patients and classified in the family Paramyxoviridae under the new genus
Henipavirus. Its genome encodes six structural proteins: the nucleocapsid (N) protein,
phosphoprotein (P), matrix (M) protein, fusion (F) protein, glycoprotein (G), and large (L)
protein.
Nipah virus glycoprotein G has a globular head domain formed of a six-bladed beta sheet propeller, connected via a flexible stalk domain to a transmembrane anchor. The G binds to the cellular receptors ephrin B2 are ephrin B3, mediating viral attachment. Following attachment Nipah Virus glycoprotein G undergoes a conformational change that leads to triggering of glycoprotein F which leads to membrane fusion (Biering et al, 2012).
The Nipah virus glycoprotein G is a recombinant protein expressed in mammalian HEK293 cells. It is presented as a fusion protein with a mouse Fc tag linked to the C-terminus of glycoprotein G, amino acids 71-602.
We established preliminary specifications defining acceptable ranges for the parameters indicated herein below for our Anti Nipah Virus IgM Capture ELISA kit. These parameters were tracked day-to-day, run-to-run, and operator-to-operator, over a schedule defined inhouse.
Recommended assay characteristics included absorbance of a zero concentration standard; factors which describe the calibration for each standard and statistical description of the calibration curve such as coefficient of correlation, slope and/or intercept; and recovery of results on control samples. It is important to be able to relate the specifications for a parameter to expected reliability of the result. Our in-house standard defined was r=0.990.
Thermo Fisher Scientific developed 200 TaqMan assays to detect mutations in the CFTR gene. They tested the assays on DNA samples containing known CFTR mutations and achieved high call rates and accuracy. The assays can be used to study CFTR mutations according to population frequency and can detect a variety of mutation types including small and large deletions. Example data is shown for assays detecting homopolymer regions and large deletions.
Long Acting Injectables - A New Dimension for Proteins and PeptidesMerck Life Sciences
Access the recording: https://bit.ly/2xAaMba
Abstract:
Long acting injectables (LAI) have been around for decades for the delivery of small molecules and peptides to treat chronic and site-specific diseases. However, when it comes to more sensitive biological therapeutics the classical polylactide and polylactide/glycolide based systems suffer from several limitations (e.g. uncontrolled release kinetics, in situ pH drop, protein degradation) making them unsuitable. The SynBiosys® biodegradable polymeric microparticle technology combines all the features required for LAI formulations for biologics. In two case studies we will showcase sustained release formulations for peptides and proteins and demonstrate their potential via extensive in vitro and in vivo characterization.
Diese Broschüre beschreibt die Einzigartigkeit dieses innovativen und perfekt designten qPCR Realtime-Cyclers. Welcher qPCR-Cycler bietet sonst noch 120 optische Kanäle, die gleichzeitige Abarbeitung von TaqMan und Hybridisierungssonden, 7plex-Analysen, und das Alles bei einer thermischen Uniformität von 0,05°C? Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
1. Researchers developed a high-throughput bead-based assay using flow cytometry to measure IgG titers in bacterial broth cultures.
2. The assay uses protein A-coated beads incubated with bacterial broth samples and a fluorescent detection antibody, then reads samples on an Accuri C6 flow cytometer.
3. Testing showed the assay has a linear range of 2ng/mL to 64ug/mL IgG, and results matched reported titers for known high and low IgG-producing bacterial strains. The assay is suitable for screening bacterial mutant libraries.
Protein microarrays allow the immobilization and detection of large numbers of proteins on small surfaces. Three key steps in the protein microarray workflow are printing, surface selection, and imaging. Optimizing the printing process is important to minimize contamination between samples. The Omnigrid and Microgrid systems can print contact-style onto 3D substrates with controls to reduce surface damage. Multiplexed protein microarrays on plates allow high-throughput screening by testing many samples in parallel. NovaRay imaging supports multiple array formats and wavelengths for detection. An example experiment showed specific and reproducible detection of target proteins in individual wells of a multiplexed plate with no carryover between wells.
Sequential Automation of RNA and DNA preps on the same QIAcube instrumentQIAGEN
Automation of QIAGEN spin-column kits on the QIAcube saves valuable time and ensures standardized results. Since the same QIAcube may be used by multiple researchers for different applications, cross-contamination between samples and preparation technologies must be avoided (e.g., when nucleases are used). The unique instrument design and features minimize contamination between sequential preps, allowing both RNA and DNA preps to be performed on the same instrument. To show the process safety and robustness, we performed alternating automated RNA preps (requiring a DNase step) and DNA plasmid preps (requiring an RNase step). The preps were sequentially performed on the same QIAcube instrument using the RNeasy® Mini Kit and the QIAprep® Spin Miniprep Kit, respectively.
Independently, we performed a series of manually processed preps to compare with the automated preps. RNA and DNA quality and yields were similar between the two methods, showing the absence of carryover of nucleases.
Accurate quantitation of microRNA is critical for pharmacological and diagnostic studies. A multiplexed bead-based method allows simultaneous generation of standard curves for multiple microRNAs, reducing reagents and labor compared to other methods. The method produced standard curves for 9 microRNAs simultaneously using a single 23-well plate in 4 hours, with coefficients of variation below 5% and the ability to test 74 tissue samples on the same plate.
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.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
The document describes the phage display technique, which involves genetically fusing a protein to the coat protein of a bacteriophage virus. This allows for the display of large protein and peptide libraries on the surface of the phage. The technique has various applications, including epitope determination, enzyme engineering, organ targeting, and isolating allergens. It involves creating a vector fusion, binding and selecting phages to a target, washing, eluting bound phages, and amplifying the process through bacterial infection.
QIAcube® RNA isolation from stool samples using the RNeasy® PowerMicrobiome® ...QIAGEN
This application note demonstrates that RNA is extracted efficiently from stool samples using the RNeasy PowerMicrobiome Kit and the QIAcube system. Furthermore, the RNA isolated with the RNeasy PowerMicrobiome Kit and the QIAcube system is compatible with downstream applications.
NEBNext Ultra DNA for Illumina NGS (ChIP-seq and HLA)_Biomek FXP Automated Wo...Zachary Smith
This document describes the automation of the NEBNext Ultra DNA Library Preparation Kit for Illumina on the Beckman Coulter Biomek FXP liquid handler. The automation allows preparation of up to 96 individually indexed DNA sequencing libraries in approximately 4.5 hours. An intuitive user interface allows specification of sample number and size selection options. Quality control analysis showed the automated libraries had over 1.4 million reads per sample, low contamination, and average insert sizes of 400 bp, demonstrating the method produces high quality libraries.
Assessment of Y chromosome degradation level using the Investigator® Quantipl...QIAGEN
Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.
Ernesto Picardi – Bioinformatica e genomica comparata: nuove strategie sperim...eventi-ITBbari
Bioinformatica e genomica comparata: nuove strategie sperimentali e computazionali per la produzione e analisi di dati NGS finalizzati a sviluppare processi e prodotti innovativi per la salute dell’uomo, l’ambiente e l’agroalimentare.
A Turn-Key Flow-Through-Mode Purification Process to improve Quality and Safe...Merck Life Sciences
In this webinar, you will learn:
Intensified plasma Immunoglobulin purifications
Scalable process development with latest technologies
Improved safety and quality of plasma IgG meeting required quality attributes
Detailed description:
Plasma-derived immunoglobulins (IgG) are essential medicines that are in worldwide shortage. How to develop optimized processing steps for robust and efficient manufacturing has been a constant goal, to make the most out of the precious plasma raw material.
In this study, we present a worse-case equivalent of plasma intermediate, explore various process steps along the fractionation flow, including flow-through-mode chromatography, affinity chromatography, virus inactivation steps and removal of solvent/detergent, single-pass TFF (SPTFF), clarification, and aseptic filtration, to establish a robust, easy-to-operate, readily scalable plasma IgG process with over 99% purity, depletion of IgA, isoagglutinin, and thrombogenic markers, meeting the commonly required 20% concentration for subcutaneous IgG infusion. Such solutions would be appropriate for various IgG intermediates which help to improve the global supply of immunoglobulins.
Next-generation sequencing (NGS) has revolutionized the way we analyze diseases and commercial outfits such as Illumina, Helicos, QIAGEN and Pacific Biosciences have made significant contributions. In addition, the launch of direct-to-consumer genetic testing solutions has dramatically changed the way consumers access genomics data. Until a few years ago, the cost of sequencing was a major bottleneck. Recent developments have reduced the cost from thousands of dollars to a couple of cents per megabase. When did these changes start? What were the changes in the commercial sector in the last 15 years? This infographic is a timeline of the NGS commercial marketplace.
Breaking the Status Quo: Using Mass Spectrometry to detect Host Cell ProteinsMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3b3Tbcd
Measurement of host cell proteins is vital to ensuring a biotherapy's purity and a patient's safety. Biotherapies treat diseases with products produced by living organisms, as a result, host cell components must be characterized and controlled. We'll review new methods within product characterization for detection.
Trace amounts of host cell proteins can be present after the production and purification of any biopharmaceutical. Detection of these species requires highly specific techniques to accurately quantify even low levels of contamination. Host cell protein impurities, present at PPM-levels in biotherapies, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. Their complex and diverse nature makes them challenging to detect or monitor. With acceptance criteria for host residual DNA usually set at a very low level (often =1.0 pg of DNA per mg of drug substance), effective removal techniques and sensitive methods of detection are critical.
Antibody-based techniques, like the enzyme-linked immunosorbent assay (ELISA), have been used to assess the HCP load of biotherapeutics before and after process changes. However, these techniques do not necessarily detect qualitative changes in the HCP population. In this webinar, we will discuss how mass spectrometry (MS)-based approaches coupled with ELISA methods help detect qualitative and quantitative differences in HCP populations.
In this webinar, you will learn:
• Comprehensive HCP ID and semi-quantitation
• HC agnostic process
• Creation of process specific database
• Differential clearance of specific HCPs throughout purification steps
• Monitoring of problematic species e.g. immunogenic (PLBL2), lipases and proteases
• Explanation about why 90% of BLAs filed included this HCP MS data
Digital PCR for soybean GMO detection on the OpenArray Platform: a case study...Thermo Fisher Scientific
This document describes how digital PCR on the TaqMan OpenArray platform can be used as a sensitive tool for detecting genetically modified organisms (GMOs). [The summary describes:]
1) Custom TaqMan assays were designed to differentiate between GMO and wild-type soybean DNA. 2) The assays were validated for specificity using digital PCR. 3) Spike-in experiments were conducted adding GMO DNA at levels as low as 0.01% to wild-type DNA, and digital PCR accurately measured ratios matching estimated levels.
Monoclonal antibody (mAb) therapeutics have formed and continue to form the vast majority of biopharmaceutical company pipelines today with a number of remarkable commercial successes. The advent of mAbs as therapeutics has been greatly aided by a process platform approach that has enabled rapid development and manufacturing for this class of drugs.Downstream process platforms for mAbs first evolved over a decade ago and have had a significant impact on the time and resources spent in process development. This chapter describes some of the platform approaches first used in the biopharmaceutical industry and how those platforms have evolved over the last decade based on needs as well as newly available technology. We also describe the advent of next generation mAb based constructs and the creation of possible platforms for those moieties.
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.
Technology Trends in Bioprocessing PurificationMilliporeSigma
This presentation reviews current trends in bioprocessing purification and includes key considerations for continuous processing and connected polishing for monoclonal antibodies. Topics include:
• Market trends and the evolution of next-generation processes
• Intensified capture processing
• Continuous virus inactivation
• Connected flow-through polishing
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab
Validation of anti niv igm capture elisa version#1krishgen
NiV is a negative-sense, non-segmented RNA virus that was first isolated from cerebrospinal fluid of human patients and classified in the family Paramyxoviridae under the new genus
Henipavirus. Its genome encodes six structural proteins: the nucleocapsid (N) protein,
phosphoprotein (P), matrix (M) protein, fusion (F) protein, glycoprotein (G), and large (L)
protein.
Nipah virus glycoprotein G has a globular head domain formed of a six-bladed beta sheet propeller, connected via a flexible stalk domain to a transmembrane anchor. The G binds to the cellular receptors ephrin B2 are ephrin B3, mediating viral attachment. Following attachment Nipah Virus glycoprotein G undergoes a conformational change that leads to triggering of glycoprotein F which leads to membrane fusion (Biering et al, 2012).
The Nipah virus glycoprotein G is a recombinant protein expressed in mammalian HEK293 cells. It is presented as a fusion protein with a mouse Fc tag linked to the C-terminus of glycoprotein G, amino acids 71-602.
We established preliminary specifications defining acceptable ranges for the parameters indicated herein below for our Anti Nipah Virus IgM Capture ELISA kit. These parameters were tracked day-to-day, run-to-run, and operator-to-operator, over a schedule defined inhouse.
Recommended assay characteristics included absorbance of a zero concentration standard; factors which describe the calibration for each standard and statistical description of the calibration curve such as coefficient of correlation, slope and/or intercept; and recovery of results on control samples. It is important to be able to relate the specifications for a parameter to expected reliability of the result. Our in-house standard defined was r=0.990.
Thermo Fisher Scientific developed 200 TaqMan assays to detect mutations in the CFTR gene. They tested the assays on DNA samples containing known CFTR mutations and achieved high call rates and accuracy. The assays can be used to study CFTR mutations according to population frequency and can detect a variety of mutation types including small and large deletions. Example data is shown for assays detecting homopolymer regions and large deletions.
Long Acting Injectables - A New Dimension for Proteins and PeptidesMerck Life Sciences
Access the recording: https://bit.ly/2xAaMba
Abstract:
Long acting injectables (LAI) have been around for decades for the delivery of small molecules and peptides to treat chronic and site-specific diseases. However, when it comes to more sensitive biological therapeutics the classical polylactide and polylactide/glycolide based systems suffer from several limitations (e.g. uncontrolled release kinetics, in situ pH drop, protein degradation) making them unsuitable. The SynBiosys® biodegradable polymeric microparticle technology combines all the features required for LAI formulations for biologics. In two case studies we will showcase sustained release formulations for peptides and proteins and demonstrate their potential via extensive in vitro and in vivo characterization.
Diese Broschüre beschreibt die Einzigartigkeit dieses innovativen und perfekt designten qPCR Realtime-Cyclers. Welcher qPCR-Cycler bietet sonst noch 120 optische Kanäle, die gleichzeitige Abarbeitung von TaqMan und Hybridisierungssonden, 7plex-Analysen, und das Alles bei einer thermischen Uniformität von 0,05°C? Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
1. Researchers developed a high-throughput bead-based assay using flow cytometry to measure IgG titers in bacterial broth cultures.
2. The assay uses protein A-coated beads incubated with bacterial broth samples and a fluorescent detection antibody, then reads samples on an Accuri C6 flow cytometer.
3. Testing showed the assay has a linear range of 2ng/mL to 64ug/mL IgG, and results matched reported titers for known high and low IgG-producing bacterial strains. The assay is suitable for screening bacterial mutant libraries.
Protein microarrays allow the immobilization and detection of large numbers of proteins on small surfaces. Three key steps in the protein microarray workflow are printing, surface selection, and imaging. Optimizing the printing process is important to minimize contamination between samples. The Omnigrid and Microgrid systems can print contact-style onto 3D substrates with controls to reduce surface damage. Multiplexed protein microarrays on plates allow high-throughput screening by testing many samples in parallel. NovaRay imaging supports multiple array formats and wavelengths for detection. An example experiment showed specific and reproducible detection of target proteins in individual wells of a multiplexed plate with no carryover between wells.
The ArrayGradeTM FFPE RNA isolation kit provides a more effective method for isolating RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. It yields RNA of higher quantity and quality compared to other commercial kits. The RNA isolated has greater compatibility with microarray and real-time PCR applications, providing more positive results and sensitivity. This allows researchers to reliably perform gene expression profiling on archived FFPE samples to gain insights into cancer progression and other disease studies.
A next generation sequencing based sample-to-result pharmacogenomics research...Thermo Fisher Scientific
Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy numbers may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact drug efficacy. PGx studies are becoming increasingly important for precision medicine. We have developed a next generation sequencing (NGS) PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With this highly multiplexed PGx research panel we can profile a set of 136 genetic markers in 40 known PGx related genes (Table 1) and determine CYP2D6 copy number variation (CNV, Figure 1) in a single reaction using Ion Torrent™ semiconductor sequencing.
Target enrichment enables researchers to focus their next generation sequencing (NGS) efforts on regions of interest, allowing them to obtain more sequencing data relevant to their study. In-solution target capture is a method of enrichment using oligonucleotide probes directed to specific regions within a genome. Target capture can be used to enrich multiple samples simultaneously, reducing the cost per sample, while using individually synthesized probes allows researchers to construct gene panels that can be optimized over time.
Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTec...Ilya Klabukov
Illumina's technology has potential clinical applications in molecular diagnostics. Their sequencing platforms like MiSeq and analysis software like KaryoStudioDx can enable molecular cytogenetic testing and targeted gene panels. MiSeq allows scalable amplicon sequencing from hundreds to thousands of targets in 1-2 days at lower costs than Sanger sequencing. Illumina aims to submit their iScan platform and arrays for FDA approval for post-natal cytogenetic testing. Their SNP microarrays can detect more types of chromosomal abnormalities than microarrays and help characterize regions associated with disease.
A choice of population-optimized GWAS imputation grids, combined with exome, loss-of-function, ADME, and eQTL markers in one high-coverage, high-value, customizable design.
This document describes how real-time PCR can be used to validate microarray data. Real-time PCR provides a quantitative and sensitive method for confirming changes in gene expression observed in microarray experiments. The document outlines a protocol for designing and running a real-time PCR experiment to validate a specific result from a microarray experiment showing increased expression of the TNFAIP3 gene in response to TNFα treatment. Key steps in the protocol include performing reverse transcription of RNA to generate cDNA, setting up a standard curve and controls, and analyzing the real-time PCR data to calculate fold-changes in gene expression.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Improved Reagents & Methods for Target Enrichment in Next Generation Sequencing, presented by Dr Mark Behlke, Chief Scientific Officer at Integrated DNA Technologies
Detection of transgenic canola (Roundup Ready® - Monsanto)claudio iannetta
Determination of a validation protocol, based on
established European Union methods, for the detection
of transgenic canola (Roundup Ready® - Monsanto) in
seed samples using molecular techniques
VIDAS and ARCHITECT are two automated immunoassay systems for serology testing. VIDAS uses enzyme-linked fluorescent assay technology to provide high-quality on-demand results for up to 80 tests per hour. It offers a variety of routine and specialty serology tests in a single-test format. ARCHITECT uses chemiluminescent microparticle immunoassay technology to provide high productivity of up to 200 tests per hour with a 135 sample load and 25 reagent positions. Both systems provide reliable, automated testing for diagnosing various infections like hepatitis B, HIV, and toxoplasmosis.
The document describes Phase II of the ABRF Next Generation Sequencing Study which aims to establish reference data sets for evaluating DNA sequencing performance across multiple platforms and laboratories. Phase II will sequence various human and bacterial genomic samples to assess accuracy, coverage, and limits of detection using different platforms and library preparation methods. A collaboration with NIST Genome in a Bottle will provide standardized samples to the participating laboratories. The study aims to provide a resource for ongoing method development and evaluation of sequencing performance.
Long reads from nanopore sequencing provide advantages for sequencing small genomes like bacteria and viruses. Key benefits include easier genome assembly from long overlapping reads, resolution of structural variants and repeats from single long reads, and real-time analysis. Nanopore devices are scalable and can sequence genomes from viruses to plants depending on the device and number of flow cells used.
This document provides information about a plant pathogen detection kit from Norgen Biotek Corp. that allows for the isolation and detection of fungal pathogens from plant samples using PCR. The kit contains components for isolating DNA from plant tissues using spin column chromatography. It also contains master mixes for amplifying fungal DNA, as well as controls. The kit is a ready-to-use system for detecting pathogens like Aspergillus niger, Botrytis cinerea, Cladosporium cladosporioides, Penicillium sp., and others from plant samples in under 3 hours.
The document discusses marker-assisted breeding and the services provided by the Sequencing and Genotyping Platform. It outlines the steps in marker-assisted selection, from laying out seedlings and collecting samples to running analyses. It also lists the facilities and equipment available, including robotic platforms for liquid handling and DNA/RNA extraction, real-time PCR systems, capillary sequencers, and Illumina platforms for high-throughput genotyping. The platform provides support for marker-assisted breeding programs through services like whole genome sequencing, targeted resequencing, and protocol development for next-generation sequencing applications.
Genome in a Bottle - Towards new benchmarks for the “dark matter” of the huma...GenomeInABottle
The document discusses Genome in a Bottle (GIAB) and its efforts to characterize human genomes and provide reference materials and benchmarks to evaluate genome sequencing and variant calling. Specifically, it summarizes how GIAB has characterized 7 human genomes, provides extensive public sequencing data for benchmarking, and is now using linked and long reads to expand the small variant benchmark set, develop a structural variant benchmark, and perform diploid assembly of difficult regions. It also shows how new benchmarks that include more difficult regions have revealed errors in previous benchmarks and reduced performance metrics for variant calling tools.
This document summarizes a study comparing RNA sequencing (RNA-Seq) results from challenging sample types amplified using NuGEN Technologies' Ovation RNA-Seq and Ovation RNA-Seq FFPE systems. The study found that both systems produced high-quality sequencing data from as little as 500 picograms or 100 nanograms of total RNA, respectively, without requiring rRNA reduction or polyA selection. Differential expression analysis of RNA from formalin-fixed paraffin-embedded (FFPE) samples showed high concordance with matched fresh frozen samples. The results demonstrate the ability to reliably study disease using archived FFPE samples.
This document discusses clinical and consumer applications of microarrays and genotyping technologies. It provides an overview of genotyping and different technologies like PCR microarrays and SNP microarrays. It describes how microarrays are still useful despite the rise of sequencing due to their low cost, high throughput, and ability to test millions of markers. The document outlines several applications of microarrays like direct-to-consumer testing, pharmacogenetics, and clinical sequencing. It also discusses challenges and trends in these areas like global initiatives to increase genomic data sharing.
Styles of Scientific Reasoning, Scientific Practices and Argument in Science ...Elsa von Licy
The document discusses various topics related to scientific reasoning, practices, and argumentation including different styles of scientific thinking, features of scientific knowledge, and teaching and learning science. It provides examples of "crazy ideas" in science that are now accepted, examines the role of argument in science, and outlines the scientific practices and central questions of science. It also discusses developing models, planning investigations, analyzing data, and constructing explanations as key scientific practices.
Anti-philosophy rejects traditional philosophy and logic, instead embracing creativity, spirituality, and personality. It considers philosophy to be dead, kept alive artificially by analytic philosophers. The document criticizes how philosophy is currently taught and argues it has become unproductive, replacing original aims with nonsense. Anti-philosophy's goal is not to destroy philosophy but to transform its current state and avoid fundamentalism in philosophy and science.
There is no_such_thing_as_a_social_science_introElsa von Licy
This document provides an introduction and overview of the arguments made in the book "There is No Such Thing as Social Science". It begins by stating the provocative title and questioning whether the authors will take it back or qualify their position.
It then outlines three ways the term "social science" could be used - referring to a scientific spirit of inquiry, a shared scientific method, or reducibility to natural sciences. The authors argue against the latter two, methodological and substantive reductionism.
The introduction discusses how opponents may accuse the authors of being a priori or anti-reductionist, but argues that those defending social science are actually being dogmatic by insisting it must follow a scientific model. It frames the debate as being
1. White Paper
Oligo GEArrays®: The Pathway-Focused DNA
Microarray System for Every Laboratory
By Heather Fox-Brashears, George Quellhorst, Raymond Blanchard, Bill Wang and Sean Yu
SABiosciences
6951 Executive Way
Frederick, MD 21703
+1 (301) 682-9200 Phone
+1 (301) 682-7300 Fax
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heatherf@SABiosciences.net
ABSTRACT
This paper describes the technology and performance characteristics of the Oligo GEArray® DNA microarray
system. Oligo GEArrays® are an effective alternative to genome-wide microarrays, in that each array is designed
to profile expression of a focused gene panel from a particular biological pathway or disease state. The data
presented in this paper substantiates the sensitivity, reproducibility, and reliability of this innovative approach
to microarray design. The Oligo GEArray’s® flexible protocol options, ease-of-use, and adaptability to any
laboratory setting provide researchers with a robust tool for routine use of microarray technology in everyday
experiments.
2. Pathway-Focused Oligo GEArrays®
Introduction to Microarray Technology and Oligo GEArrays®
Microarray technology utilizes RNA from cells, tissue, and other biological source materials to
simultaneously determine the expression levels of many genes. Microarrays are composed of a set of distinct,
gene-specific, nucleic acid probes immobilized on a solid support. During a microarray experiment, RNA
is enzymatically converted to labeled cDNA (complementary DNA) or cRNA, and then hybridized to the
immobilized nucleic acid probe. The labeled target bound at each gene-specific spot is typically detected using
chemiluminescent, fluorescent, or radioactive methods. The signal produced at each spot is representative of the
amount of message in the original RNA sample.
Advances in DNA microarray technology have facilitated genome-wide expression profiling with
whole genome microarrays available from companies such as Affymetrix, Agilent or Illumina. While these
microarrays provide a solution for discovery-based research endeavors, the large amount of data produced from
genome-wide arrays requires the use of specialized software and daunting analysis procedures. SABiosciences
pathway-focused, lower-density Oligo GEArray® microarray system provides a more economical and productive
alternative for monitoring a focused panel of genes in a particular biological pathway or disease state. Oligo
GEArrays® are comprised of a nylon membrane support spotted with gene-specific 60-mer oligos for up to 440
different genes. Oligo GEArrays® use as little as 10 pg of total RNA, with a chemiluminescence detection method.
No special equipment is required for the Oligo GEArray® platform, facilitating its easy adoption into any
biological research laboratory. These arrays offer comparable performance to high-density microarray formats
while enabling hypothesis-driven experimental design, relevant data acquisition, and efficient data analysis.
Oligo GEArray Characteristics
The Oligo GEArray® product platform combines expertly designed pathway-specific microarrays and
advanced oligonucleotide probe design with a streamlined labeling, hybridization, and detection protocol
(Figure 1). The distinct advantages of the Oligo GEArray® system are outlined in Table 1. With the combined
benefits of the membranes, reagents, uncomplicated protocol, and the GEArray® Analysis Suite Software, the
Oligo GEArray® product line provides a complete and convenient microarray experiment package for any
laboratory.
Table 1: Features and Benefits of the Oligo GEArray® System
Focused Array Design
Lower-density (113-440 genes) arrays designed by biological pathway or disease state for relevant
data acquisition
Excellent Quality Manufacturing
Carefully-designed mRNA 3’ end biased 60-mer oligonucleotide probes
(Median distance ~160 bases from 3’ end) on a positively-charged nylon membrane with noncontact printing method (10 nL deposition volume)
Easy Target Labeling
One-tube proprietary IVT-based linear RNA amplification labeling kit using 0.1 to 3 µg of total RNA
Various Detection Method
Single color / channel chemiluminescent detection or X-ray film
Express Protocol
With the HybPlate Array format, you can easily process up to 32 arrays in one day
Straightforward Data Analysis
Software
Web-based software available by subscription
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3. SABiosciences
HybPlate
Express
Protocol
(Single Day
Experiment)
Standard
HybTube
Protocol
(Two Day
Experiment)
3 hours
How It Works
3 hours
3.5 hours in
Thermoshaker
Overnight in
Hyb Oven
0.75 hours
1.5 hours
0.5 hours
0.5 hours
Preparation of Labeled cRNA with TrueLabelingAMP™ 2.0
Start with as little as
0.1 - 3.0 µg of Total RNA
Amplify
Labeled cRNA
Target
Hybridization and Washing
Simply add cRNA target to the
GEArray and follow the easy
hybridization and wash steps.
ce
urfa
yS
Arra
Signal Development
Detection is as simple as
developing a Western Blot
using chemiluminescence.
Image Aquisition
Use a CCD-camera system to
capture chemiluminescence.
You can also use X-ray film
and a standard desktop scanner.
®
Analysis with GEArray Expression Analysis Suite
Auto-align the gene table for your arrays to the captured
image, select your analysis parameters, and go!
Graphical and tabulated differential gene expression
data is at your fingertips.
Figure 1: Outline of the Oligo GEArray® experimental procedure
Oligo GEArrays are composed of 113-440 gene-specific 60-mer oligos that have been rigorously evaluated
for specificity, sequence complexity, secondary structure, melting temperature, GC content, and distance to 3’
end of transcript. The oligo probes are immobilized on a positively charged, three-dimensional nylon membrane.
The nylon membrane matrix offers distinct advantages over impermeable surfaces such as chips and glass slides,
including larger probe immobilization and increased hybridization and detection area. When combined with
an optimally formulated hybridization solution and sensitive chemiluminescent detection, the Oligo GEArray
membrane maximizes both hybridization efficiency and signal generation.
The choice of two different target labeling kits is an integral part of the Oligo GEArray platform. The
TrueLabeling-AMP™ 2.0 linear amplification and labeling kit is a universal target labeling method specifically
designed to convert total RNA to amplified and labeled cRNA (Step 1, Figure 1). Based on standard in vitro
transcription based methods, the TrueLabeling-AMP™ 2.0 procedure requires fewer enzymatic reactions, only
one purification step, and a significantly shorter protocol as compared to traditional procedures. The biotinylated
cRNA target contains the same 3’ biased sequences as the gene-specific oligonucleotides printed on the array to
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4. Pathway-Focused Oligo GEArrays®
enhance efficient hybridization and maximize signal. Only 0.1 to 3 µg of total RNA starting material is required
for the TrueLabeling-AMP 2.0 kit to produce sufficient cRNA for hybridization. When using a consistent amount
of cRNA in the hybridization step, differing amounts of total RNA can be utilized to produce similar microarray
results (Figure 2). The TrueLabeling-PicoAMP™ two-round amplification and labeling kit is designed for very
small samples with as little as 100 picograms of RNA (10 cells), and provides comparable results to the standard
one-round True Labeling-AMP 2.0. In Figure 3, a comparison is shown between microarray results with varying
amounts of input RNA in the two-round TrueLabeling-PicoAMP™ kit, and results obtained with the one-round
True Labeling-AMP kit. The TrueLabeling-PicoAMP™ kit offers an excellent solution for laboratories interested
in performing microarray studies on sources of limited RNA, such as laser capture microdissection (LCM),
Formalin-Fixed Paraffin Embedded (FFPE), or Fine Needle Aspiration Biopsy (FNAB) samples.
Normalized Signal Intensity (SI)
4.00
Log(SI) from 100ng RT-IVT input
3.00
2.00
1.00
0.00
-1.00
-2.00
-3.00
-4.00
y = 1.0102x - 0.1532
2
R = 0.9738
-5.00
-6.00
-6.00
-4.00
-2.00
0.00
2.00
Log(SI) from 3ug RT-IVT input
4.00
Figure 2: Biotinylated cRNA synthesized from different amounts of total RNA produce similar array results. A constant amount (2 µg) of
cRNA generated from differing amounts of total RNA (listed above) was hybridized to separate Oligo GEArray Human Tumor Metastasis
Microarrays (HybTube format, catalog number OHS-028) for 18 hours at 60oC. Hybridization, washing, and chemiluminescent detection
were performed as outlined in the User Manual. A good correlation was observed between the normalized signal intensities obtained with
different total RNA inputs.
1.50
Normalized Signal Intensity (SI)
Log(SI) from 500pg total RNA
1.00
0.50
0.00
-0.50
-1.00
y = 0.9064x + 0.0513
2
R = 0.9336
-1.50
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
Log(SI) from 500ng total RNA
Figure 3: Similar microarray performance is observed with 500 pg total RNA starting material with the TrueLabeling-PicoAMP™ kit as
compared to 500 ng total RNA starting material with the TrueLabeling-AMP™ 2.0 kit. Human XpressRef™ Total RNA (500 pg) was used in
the TrueLabeling-PicoAMP™ procedure as outlined in the User Manual. The 1-round TrueLabeling-AMP™ 2.0 amplification and labeling
procedure was performed as outlined in the User Manual with 500 ng of the Human XpressRef™ Total RNA. For the hybridization step, 2 µg
of each cRNA target was hybridized to separate Oligo GEArray® Human Tumor Metastasis Microarrays (HybTube format, catalog number
OHS-028). Hybridization with 2-round cRNA produced a similar pattern and comparable percent present call to the 1-round cRNA. A good
correlation in normalized signal intensity was observed between the two labeling methods.
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5. SABiosciences
The Oligo GEArray® hybridization protocol, array formats, and detection methods also offer flexibility
and processing options for every laboratory. The traditional HybTube array format is designed for use with the
standard rotisserie-style hybridization ovens. The HybPlate format is ideal for high-throughput applications,
allowing the processing of up to 32 arrays in one day with the use of SABiosciences' specialized GEArray®
Express Thermoshaker. The GEArray® chemiluminescent detection system, optimized for use with the nylon
membrane matrix, offers excellent sensitivity, wide dynamic range (up to 4 orders of magnitude), and versatile
image acquisition options. Chemiluminescent detection provides comparable or better sensitivity and signal as
compared to radioactive detection methods, without the worry of radioactive waste disposal and precautionary
laboratory measures. The array images can be captured using a cooled CCD camera (for best results) or simply
with X-ray film and the use of a desktop scanner. When used with autoradiography film, chemiluminescent
detection methods are generally more sensitive than radioactivity. Array data analysis is facilitated through the
use of SABiosciences' GEArray® Expression Analysis Suite web-based software. This subscription-based software
offers automated, array-specific analytical functions as well as various analysis tools including scatter plots and
hierarchical clustering.
The Oligo GEArray® is a user-friendly, efficient, and focused DNA microarray tool that offers superior
pathway-focused solutions for gene expression studies. The straightforward protocol includes a cRNA
amplification and labeling portion that can be completed in as little as 2.5 hours. In addition, the protocol
provides flexible hybridization options that include a 1-day express procedure (HybPlate format only), and a
simple and safe chemiluminescent detection. Oligo GEArrays® provide a convenient, cost-effective, sensitive, and
pathway-focused alternative to genome-wide microarrays.
Oligo GEArray Performance: Sensitivity, Dynamic Range, Reproducibility, and Reliability
Typical performance parameters for microarray validation include sensitivity and dynamic range,
reproducibility, and reliability (compatibility with real-time PCR results). The Oligo GEArray® system affords
excellent sensitivity, a wide dynamic range (up to 4 orders of magnitude), robust reproducibility, and good
correlation with real-time PCR experiments. Oligo GEArrays® offer similar or better performance as compared to
popular genome-wide array systems (Table 2).
Table 2: Typical System Performance (*based on published Affymetrix technical notes)
Oligo GEArray®
Affymetrix™ GeneChip®*
Sensitivity
As low as 10 fM synthetic target
125 fM
Linear Dynamic Range
3 orders of magnitude
3 orders of magnitude
Specificity
60-mers provide superior specificity as
compared to shorter probes
25-mer probes provide less sensitivity that
60-mer or 70-mer probes
Reproducibility
~ 10% CV
10-15% CV
Sample Input
10 pg – 3 µg total RNA
At least 100 ng RNA
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6. Pathway-Focused Oligo GEArrays®
To address the qualities of sensitivity and dynamic range, known quantities of labeled synthetic
RNA target were spiked into a labeled human RNA sample for the performance of a series of hybridization
experiments (Figure 4). The Oligo GEArray® exhibits a low-limit of detection in the 10 fM range, as demonstrated
by detection of the target at a spiking ratio of 1:20,000,000. This performance level indicates that the Oligo
GEArray® is an excellent tool for studying low abundance transcripts (less than 10 copies per cell); however,
the wide linear dynamic range allows for the simultaneous, accurate detection of both high and low abundance
messages.
Performance determined by titration of synthetic target
100000
Raw Intensity
R2 = 0.9969
10000
1000
100
noise level
10
0.00
0.01
0.10
1.00
10.00
100.00
Luciferase cRNA (pM)
Figure 4: The Oligo GEArray exhibits a high level of sensitivity and wide linear dynamic range. A firefly luciferase in vitro transcript
was converted to labeled cRNA target using the TrueLabeling-AMP method. Different known amounts of the target (10 fM to 10 pM) were
mixed with a constant amount of labeled cRNA target generated from XpressRef™ Human Universal Reference Total RNA (GA-004). Each
mixture of labeled cRNA target was hybridized to replicate Oligo GEArray® Human Microarrays. The luciferase gene raw signal intensity
was plotted versus the quantity of labeled luciferase cRNA. These results demonstrate that the Oligo GEArray® can detect as little as 10 fM
labeled target with a linear dynamic range of four orders of magnitude (from 0.01 pM to 10 pM).
Oligo GEArrays® are manufactured utilizing an advanced, non-contact printing technology that ensures
excellent array print quality and reproducibility. Technical replicates within an experiment generally yield
coefficient of variation values (CVs) of less than 10%. Figure 5 illustrates a representative comparison between
the results of duplicate Oligo GEArrays®. The results demonstrate a high degree of correlation between the data
sets; thus indicating that the Oligo GEArray® system is a robust, reproducible method to reliably detect small, yet
biologically significant changes in gene expression.
100000
Hybridization 1
R2 = 0.9939
10000
1000
100
100
1000
10000
Hybridization 2
100000
Figure 5: The Oligo GEArray® exhibits excellent array-to-array reproducibility. XpressRef™ Human Universal Reference RNA (GA-004)
was converted to labeled cRNA target using the TrueLabeling-AMP™ Linear RNA Amplification Kit (GA-010). Equal amounts (2 µg) of
amplified product were hybridized to replicate Oligo GEArray® Human Microarrays. The Chemiluminescent Detection Kit (D-01) was used
to detect the presence of labeled target on the arrays. The raw intensity values for each gene from one array were plotted against those
values from the second array, and the data were fit to a straight line having a correlation factor (R2) of 0.9939.
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A complete gene expression profiling experiment requires the validation of microarray results by
real-time PCR (or an alternative method). Verification by real-time PCR should yield a closely matched gene
expression profile (70-90% agreement) to that observed with the microarray in order to regard the data as
reliable. Figure 6 illustrates the results of a gene expression profiling experiment in which the cancer-related
gene expression was compared between samples from two different breast cancer cell lines. The results obtained
with the Oligo GEArray® were confirmed by real-time PCR analysis of 84 genes using SABiosciences' RT2 RealTime™ Primer Sets. Using a fold-change threshold of 1.3, the relative gene expression changes for the cell samples
using the Oligo GEArray® and real-time PCR exhibited greater than 70% agreement, indicating a high degree of
reliability in the Oligo GEArray® results.
A
MCF7
B
2.5
MDA-MD-231
2.0
RT2 Real-Time™ PCR
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-1.5
-1.0
-0.5
0
0.5
1.0
Oligo GEArray® Human Cancer Microarray
1.5
Figure 6: The Oligo GEArray® demonstrates good correlation with real-time PCR results. RNA (3 µg) from two different of human breast
cancer cell lines, MCF7 and MDA-231, was converted to labeled cRNA target with the TrueLabeling-AMP™ RNA Linear RNA Amplification
Kit (GA-010). Triplicate samples of labeled target (6 µg) were hybridized to separate Human Oligo GEArray® Cancer Microarrays (OHS-802).
Sample array images are shown in Panel A. RNA (7 ng) from each cell line was also converted into first strand cDNA. Each cDNA sample
was used in multiple PCR reactions (n=5) of 84 genes represented by the microarray using RT2 Real-Time™ PCR Primer Sets. A ratio of relative gene expression between the two cell lines was calculated for each gene. The results determined with the two different experimental
methods are plotted against one another in Panel B. This result demonstrates a good agreement between the data sets indicating that the
GEArray® results are sufficiently reliable when compared to real-time PCR.
Discussion and Product Information
The Oligo GEArray® DNA microarray system offers an exceptional research solution for investigators
interested in studying gene expression profiles related to specific pathways or disease states. The pathwayfocused approach to array design, combined with the excellent performance characteristics described above,
affords researchers an effective and reliable tool for human, mouse, or rat gene expression discovery and
screening applications. Coupled with the straightforward procedure, the web-based data analysis software
and flexible protocol options allow the easy integration of the Oligo GEArray® platform into any laboratory.
SABiosciences offers a variety of products complimentary to the Oligo GEArray® line, including an ArrayGrade™
Total RNA Isolation Kit, RT2 Real-Time™ Primer Sets, instrument-specific master mixes, and other real-time
PCR reagents. A list of available microarrays is shown on the next page. Visit www.SABiosciences.com for more
information and a complete product catalog.
Email support@SABiosciences.com
Web www.SABiosciences.com
8. Pathway-Focused Oligo GEArray® Microarrays.
For a complete listing, visit www.SABiosciences.com/ArrayList.php
Pathway / Topic Focus
Alzheimer’s Disease
Angiogenesis
Apoptosis
Atherosclerosis
Autoimmune and Inflammatory Response
Breast Cancer Biomarkers
Cancer
Cancer PathwayFinder™
Cardiovascular Disease Biomarkers
Cell Cycle
Cell Surface Markers
Chemokines and Receptors
Common Cytokines
Diabetes
DNA Damage Signaling Pathway
Endothelial Cell Biology
Extracellular Matrix and Adhesion Molecules
Genome Stability / DNA Repair
Hematology/Immunology
Hematopoietic Stem Cells and Hematopoiesis
HIV Infection and Host Response
Hypoxia Signaling Pathway
Inflammatory Cytokines and Receptors
Innate and Adaptive Immune Responses
Insulin Signaling Pathway
Interferon α,β Response
JAK / STAT Signaling Pathway
Neurogenesis and Neural Stem Cells
Neuroscience-1 Ion Channels and Transporters
Neurotransmitter Receptors and Regulators
Neurotrophins and Receptors
NFkB Signaling Pathway
Notch Signaling Pathway
Nuclear Receptors Coregulators
Obesity
Osteogenesis
p53 Signaling Pathway
PI3K-AKT Signaling Pathway
Prostate Cancer Biomarkers
Signal Transduction PathwayFinder™
Stem Cell
Stress Response to Cellular Damage
T-cell and B-cell Activation
TGFβ / BMP Signaling Pathway
Th1-Th2-Th3
Toll-Like Receptor Signaling Pathway
Toxicology and Drug Resistance
Tumor Metastasis
WNT Signaling Pathway
HybPlate Format
Human
EHS-057
EHS-024
EHS-012
EHS-803
EHS-402
EHS-802
EHS-033
EHS-037
EHS-020
EHS-021
EHS-015
EHS-013
EHS-042
EHS-801
888-503-3187 (USA)
301-682-9200
888-465-9859 (USA)
301-682-7300
support@SABiosciences.com
www.SABiosciences.com
EMM-024
EMM-012
Rat
ERN-024.2
ERN-012.2
EMM-803
EMM-033
ERN-020
EMM-022
EMM-021
EMM-015
EMM-013
EHS-032
EHS-011
EMM-032
EMM-011.2
EMM-052
EHS-039
EHS-404
EMM-039
EMM-404
EHS-060
EMM-060
EHS-025
EHS-059
EHS-056
ERN-015.2
EMM-059
EHS-026
EHS-027
EHS-058
EHS-403
EHS-014
EHS-405
EHS-035
EHS-034
EHS-018.2
EHS-401
EHS-028
EHS-043
Contact us for more information on
Oligo GEArrays®:
Phone:
Fax:
Email:
Web:
Mouse
HybTube Format
ERN-032
ERN-011.2
ERN-404
ERN-060
ERN-031
EMM-026
ERN-026
EMM-058
ERN-058
EMM-014
EMM-405
ERN-014.2
EMM-034
EMM-018.2
EMM-401
EMM-043
ERN-401
ERN-029
Human
OHS-057
OHS-024
OHS-012
OHS-038
OHS-803
OHS-402
OHS-802
OHS-033
OHS-037
OHS-020
OHS-055
OHS-022
OHS-021
OHS-029
OHS-015
OHS-013
OHS-042
OHS-801
OHS-054
OHS-051
OHS-032
OHS-011
OHS-052
OHS-030
OHS-039
OHS-404
OHS-036
OHS-060
OHS-031
OHS-025
OHS-059
OHS-056
OHS-026
OHS-027
OHS-058
OHS-403
OHS-014
OHS-405
OHS-053
OHS-035
OHS-034
OHS-018.2
OHS-401
OHS-028
OHS-043
Mouse
Rat
OMM-024
OMM-012
OMM-038
OMM-803
ORN-024.2
ORN-012.2
OMM-033
OMM-037
OMM-020
OMM-055
OMM-022
OMM-021
OMM-023
OMM-029
OMM-015
OMM-013
OMM-042
ORN-020
ORN-022
ORN-021
ORN-029
ORN-015.2
ORN-013
OMM-054
OMM-032
OMM-011
OMM-052
OMM-030
OMM-016
OMM-039
OMM-404
OMM-036
OMM-060
OMM-031
OMM-025
OMM-059
OMM-056
OMM-017
OMM-026
OMM-027
OMM-058
ORN-032
ORN-011.2
OMM-014
OMM-405
OMM-019
OMM-053
OMM-035
OMM-034
OMM-018.2
OMM-401
OMM-028
OMM-043
ORN-014.2
ORN-404
ORN-060
ORN-031
ORN-025
ORN-026
ORN-027
ORN-058
ORN-035
ORN-034
ORN-018
ORN-401
ORN-028
ORN-043
Free Training!
Attend one of our Web Seminars to learn
more about Oligo GEArray® Methods:
www.SABiosciences.com/seminarlist.php