The field of microbiological research is faced with challenges surrounding the lack of quality, completeness, authenticity, accuracy, and traceability in publicly available reference genomes. These issues arise from the use of non-authenticated cultures and older sequencing and analysis technologies, as well as the absence of standardized methodologies for sequencing and assembling reference genomes. As a result, researchers require reliable genomic information tied to authenticated, fully characterized materials with known and reliable provenance. To address these challenges, the authors of this white paper from ATCC surveyed the status of ATCC bacterial genome sequences in public databases and implemented a genome sequencing workflow designed to provide reference-quality whole-genome sequences derived from authenticated ATCC materials. Their results demonstrated a significant lack of representation of ATCC strains with complete genome sequences available in public databases. The authors then developed and applied a standardized end-to-end sequencing and assembly workflow for producing reference-quality genome sequences, aiming to improve the quality of reference genome sequences and facilitate accurate interpretation of research results.

Human monkeypox virus (hMPXV) is a zoonotic viral disease endemic to central and western Africa, causing a smallpox-like disease in humans. Rapid and accurate detection of hMPXV during early stages of infection is essential for containment and proper treatment. While culture-based approaches can be used to detect hMPXV, they are time-consuming and labor-intensive, making PCR-based methods a more attractive alternative for rapid and highly sensitive screening. To address the need for high-quality reference materials in PCR assay development and validation, ATCC designed and developed a quantitative synthetic molecular standard for hMPXV, containing biomarkers for Clade I (Congo Basin) and Clade II (West African). The standard was authenticated via next-generation sequencing and quantified using droplet digital PCR via published quantitative PCR assays. The synthetic DNA construct proved compatible with all assays tested, demonstrating its potential as a control for assay development, verification, and validation.

This white paper from ATCC discusses the development and validation of this standard.

The increasing accessibility of next-generation sequencing (NGS) technologies has greatly influenced microbiome analyses, with applications in clinical, diagnostic, therapeutic, industrial, and environmental research. However, the complexity of 16S rRNA and metagenomic sequencing analysis presents significant challenges due to biases introduced during sample preparation, DNA extraction, PCR amplification, library preparation, sequencing, or data interpretation. A primary challenge in assay standardization is the limited availability of reference materials. To address these biases and provide a measure of standardization within microbiome research and applications, ATCC has developed a set of mock microbial communities, which includes lyophilized whole cells or genomic DNA, for use as NGS Standards in microbiome research. These standards mimic mixed metagenomics samples and comprise fully sequenced, characterized strains selected based on select phenotypic and genotypic attributes. In collaboration with ATCC, One Codex has developed a data analysis module that provides simple output in the form of true-positive, relative abundance, and false-negative scores for 16S rRNA community profiling and shotgun metagenomic sequencing.

This white paper from ATCC discusses the development and evaluation of the NGS standards for use in various applications, such as method optimization and data interpretation.

Life-extending targeted treatment options are emerging at an accelerated pace, but cancer cases across the world continue to rise. And unfortunately, the number of clinical oncologists available to treat these cancer patients is declining. Additionally, it is nearly impossible for clinicians to keep up with the volume and complexity of new genetic discoveries and treatment options. Without specialized technology, clinicians are limited in their usage of this data. As a result, GenomOncology developed a suite of solutions that intakes the extensive, evolving cancer-related information, and outputs data-driven intelligence.

In this on-demand webinar from GenomOncology, their vice president of product innovation discusses how GenomOncology has enabled push notifications within their platform, and how clinicians can customize the cadence, type, and form of communication they want to receive from GenomOncology, streamlining cohort identification, in-house clinical trial matching, and knowledge monitoring.

Analytical development groups need to consider FDA guidelines when developing quantitative, validated assays. Researchers in these groups need quantitative assays using inherently complex sample types, often with limited material available for analysis. While ELISAs have the advantages of sensitivity and specificity, matrix effects can occur when analyzing complex sample types like tissue homogenates. In addition, custom development of sandwich ELISAs can also be challenging owing to the need to identify and validate a pair of two antibodies rather than just a single antibody reactive against the target of interest.

Overestimation of analyte molecules due to crossreactivity is still a problem with ELISA. ELISA lacks the separation profiles needed to characterize off-target binding and protein isoforms, like full-length and cleaved protein targets, or non-functional and enzymatically activated prodrugs. ELISAs also use capture antibodies to bind sample analytes directly in the presence of the sample matrix which can increase the matrix effect.

This application note from Bio-Techne shows that Simple Western analysis of human endothelial nitric oxide synthase (eNOS) in brain whole tissue lysate is less susceptible to matrix effect than a leading commercial eNOS ELISA kit, allowing for more accurate quantification in brain tissue homogenates.

The reprogramming of human induced pluripotent stem cells (iPSCs) has revolutionized regenerative medicine, but gaining an accurate and quantitative understanding of signaling networks that regulate stem cell differentiation is crucial and remains a daunting challenge. The complexity of signaling networks stems from the many proteins and phosphorylated protein isoforms involved, and researchers are often challenged with limited material for analysis.

Traditional methods like western blot lack throughput, multiplexing ability, detection sensitivity, and reproducible quantification needed to gain a complete understanding of stem cell biology. Simple Western is an advanced capillary immunoblotting platform that addresses the limitations of current methods for protein expression and cell signaling pathway analysis in stem cells for regenerative medicine.

This application note from Bio-Techne shows how the automated western blot analysis of Simple Western can be used to monitor iPSC differentiation into the primary germ layers of human embryos, applying high-sensitivity multiplex assays to reveal and quantify changes in the expression of factors that regulate stem cell differentiation.

Immuno-oncology has created revolutionary therapies for some, but the full promise of immuno-oncology has yet to be realized. Among the top approaches for immunotherapies are altering the tumor microenvironment to prevent immunosuppression, blocking immune checkpoint inhibitors, and reprogramming immune cells to target antigens presented on cancer cells. Despite the diversity of these approaches, a commonality is a need for protein analytical methods that preserve precious samples, while also providing assay robustness in terms of sensitivity, reproducibility, and quantification.

To realize the full potential of immuno-oncology, ProteinSimple, a Bio-Techne brand, provides next-generation protein analytical tools to uncover the role of proteins in cancer and provide novel approaches to develop and analyze protein-based therapeutics. Simple Western is a capillary-based automated immunoassay for the characterization of proteins in complex mixtures. Unlike traditional western blot, which is time-consuming and often irreproducible, Simple Western provides highly quantitative and reproducible protein expression data in a fraction of the time (as little as three hours). Single-Cell Westerns on Milo perform western analysis on ~1,000 individual cells at a time, providing unparalleled insight into population heterogeneity.

This application note from Bio-Techne shows how Simple Western and Single-Cell Western can contribute to three important branches of immuno-oncology: angiogenesis of the tumor microenvironment, immune checkpoint inhibition, and chimeric antigen T-cell (CAR T-cell) therapy.

The enzyme-linked immunosorbent assay (ELISA) is considered the gold standard of immunoassays available today. It is a popular plate-based method for detecting and quantifying peptides, proteins, antibodies, or hormones, and is used in a wide range of both research and applied settings, including clinical applications, such as the detection of viral infections.

This e-book from Integra consists of educational articles that discuss how ELISAs work, the materials required to perform them, and their limitations in comparison to other immunoassays, such as western blot, as well as application notes and customer testimonials demonstrating how Integra solutions can improve ELISA throughput and success.

The polymerase chain reaction (PCR) is a key life sciences technique. It has been used in molecular biology — including molecular diagnostics — for many years, and several different types, such as RT-PCR, qPCR, vPCR, and ddPCR, have been developed over time. Today, PCR is a vital tool for the detection of pathogens, such as the SARS-CoV-2 virus, and is essential for genotyping and NGS library preparation. However, PCR is well known for being difficult to run successfully and several parameters must be considered when planning the PCR protocol.

This e-book from Integra consists of educational articles that discuss how PCR works and considerations for performing effective PCR reactions, as well as application notes and customer testimonials that demonstrate how Integra solutions can help to improve laboratory throughput and PCR success.

Overcoming high dead volume to save precious and expensive reagents is an important goal for laboratories. The assumption that only pipetting from single tubes guarantees the lowest possible dead volume holds up well. However, even with automated single-channel pipettes, this is not an option when high throughput is required. Integra has developed novel divided reservoirs to reduce reagent wastage when using the Voyager adjustable tip spacing pipette.

This application note from Integra demonstrates how reagent transfer can be fully automated with high accuracy and low dead volume by mounting the Voyager adjustable tip spacing pipette onto the Assist Plus pipetting robot, providing cost- and time-saving benefits when preparing master mixes for multiplex qPCR using a divided reservoir.

Automating qPCR master mix preparation is often considered extremely tedious, due to the need to constantly change program set-up based on varying master mix volumes and sample numbers. However, the D-One single-channel pipetting module for the Assist Plus pipetting robot simplifies the programming of automated qPCR master mix preparation protocols down to a single step. The D-One individually reaches tubes of different qPCR master mix components, while the Vialab program takes care of all calculations and provides automatic tip selection.

This application note from Integra verifies a SYBR Green qPCR protocol prepared directly in a divided reservoir using the D-One single-channel pipetting module for the Assist Plus pipetting robot, allowing master mixes to be pipetted into any tube or reservoir, enabling large-scale qPCR master mix preparation, with subsequent distribution to plates with multichannel pipettes to speed up the process.

Fragment analysis outputs the size of DNA fragments in a sample using capillary electrophoresis, during which negatively charged DNA molecules are separated by size as they migrate through the capillary toward a detection cell. Using multiple fluorophores allows fragments of similar sizes to be further distinguished when multiplexing. The various targets are all identified from the same sample in the same reaction, giving fragment analysis an advantage over qPCR. Fragment analysis is also cheaper and faster than NGS and allows for simpler data analysis and interpretation.

This white paper from Thermo Fisher Scientific summarizes a webinar in which the speakers discussed using fragment analysis for the multiplexed identification of viruses and bacteria, focusing on applications for artificial joint infections, inflammatory bowel disease, and improving in vitro fertilization success rates.

The live webinar was hosted by Nature Research Custom Media and sponsored by Thermo Fisher Scientific.

Cross-contamination with ambient RNA is common in single-cell RNA sequencing droplet-based platforms. In this study, we demonstrate that mouse kidney cells were less susceptible to ambient RNA contamination when analyzed with the Parse Evercode WT v2 hardware-free method than with Chromium Next GEM 3’ v3.1.

The study evaluates gene detection and cell type clusters. Evercode detected 79 percent more genes than Chromium. Annotation of the two datasets showed a large cluster of seemingly RBCs in the Chromium data, and hemoglobin RNA was found across all clusters, absent in the Evercode data. When minor tubule cells were subclustered, Evercode data showed a clear resolution of two types of collecting duct cells, while there is a single cluster in the Chromium data, further illustrating the negative impact of ambient RNA. These results demonstrate that Evercode detected more genes per cell, had substantially less ambient RNA contamination, and demonstrated higher cluster resolution.

The brain is one of the most complex and heterogeneous organs in terms of its cell types. This technical note compares two single-cell RNA sequencing technologies, the 10x Genomics Chromium Next GEM Single Cell 3’ Kit v3.1 and the Parse Biosciences Evercode WT v2, using mouse brain nuclei as a sample type.

The study evaluates gene detection, cell type recovery, and differential gene expression detection for both technologies. The Evercode WT v2 consistently outperformed the Chromium Next GEM Single Cell 3’ Kit v3.1 in gene detection at all sequencing depths, recovered similar proportions of cell types, and detected substantially higher numbers of differentially expressed genes across all cell types. The results suggest that Evercode WT v2 yields higher sensitivity and more information per cell, making it a valuable tool for single-cell RNA-seq studies in neurobiology.

For more than 10 years, BSG has been the innovator in engineered beads with differential physicochemical properties for sub-proteome separations including for example, albumin and hemoglobin removal.

This case study highlights representative journal publications whereby one or more of the BSG advantages (consumable, enrichment/depletion, functional integrity, on-bead digest) uniquely supports the research study goal. These investigations cover a range of diseases, including cancer, neurodegenerative, Covid-19, malaria, and cardiovascular.