This technical note from Beckman Coulter Life Sciences presents data from a study in which EGFR L858R standards with varying allele frequencies were spiked into plasma and isolated using either Apostle MiniMax High-Efficiency cfDNA Isolation or another extraction product, showing extraction efficiency to affect NGS results.

Due to its small size and low quantity, the main challenge for cell-free DNA analysis is to get enough cfDNA for sequencing. This results in a need to extract from larger volumes of bodily fluid, yet higher input volumes can be more difficult to manage for high-throughput sample processing.

This application note from Beckman Coulter Life Sciences demonstrates the use of the Apostle MiniMax High-Efficiency cfDNA Isolation Kit in conjunction with the KingFisher Duo Prime automated protein purification system to mitigate some of the challenges of processing large-volume samples for cell-free DNA sequencing.

As a non-invasive way to detect disease, cfDNA is extracted from blood; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. Tumor tissue is typically removed and stored as formalin-fixed, paraffin-embedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. Despite the difficulties, FFPE tissue is often used to look for cancer-associated mutations; however, it does not always correlate with the mutations seen in cfDNA.

This poster from Beckman Coulter Life Sciences presents a comparison of matched FFPE and plasma samples to determine how many mutations are seen in both tissues and where the mutational mismatches appear in the chromosome, finding that chromosomal regions have different mismatch rates and giving clues about the best chromosomal locations for biomarkers.

This white paper from Beckman Coulter Life Sciences presents data generated by the Apostle MiniMax High-Efficiency cfDNA Isolation Kit, a cell-free DNA (cfDNA) isolation reagent kit built on magnetic bead-based technology demonstrated to purify cfDNA from human plasma in both manual and automated workflows.

With recent advancements in nucleic acid-based molecular technologies, applications for precision medicine in clinical research have grown exponentially. In particular, analysis of cell-free DNA (cfDNA) for cancer detection, prognosis, and monitoring has significantly improved patients’ clinical management and outcomes.

The primary challenge with cfDNA-based liquid biopsy techniques is that cfDNA is typically found at very low concentrations in body fluids, with a range of 1 to 50 ng cfDNA from 1 mL plasma. Of the total cfDNA found in plasma, fragments originating specifically from tumors (ctDNA) can represent as low as 0.01 percent. Thus, efficient techniques for cfDNA extraction are essential for ctDNA detection in precision medicine. Two of the most commonly used cfDNA extraction techniques are spin column-based and magnetic bead-based purification.

This technical note from Beckman Coulter Life Sciences describes a study comparing a spin column-based cfDNA purification kit with a magnetic bead-based purification kit, finding the bead-based kit to significantly outperform the column-based kit and highlighting the importance of selecting an efficient cfDNA extraction method for liquid biopsy applications.

Dr. Nallasivam Palanisamy is on a journey of discovery. His destination? Finding novel biomarkers to improve the diagnosis and treatment of prostate cancer.

This customer spotlight from Beckman Coulter describes the biomarker discovery research of Nallasivam Palanisamy, associate scientist in the department of urology at Henry Ford Health, where his lab is working to understand the genetic complexity of multifocal prostate cancer and its intra- and inter-tumor heterogeneity.

Viral vectors, including adeno-associated vector (AAV), are an effective tool to deliver transgenes into the host cells. The frequency of vector transduction of targeted cells and organs can affect the efficacy, and there are two key ways to measure success: vector copy number (VCN) and transgene expression. VCN can be determined by extracting genomic DNA (gDNA) from bulk cells/tissue and utilizing quantitative PCR (qPCR) to determine the total number of viral genomes.

In the preclinical study phase, VCN and transgene expression are often tested from the same sample to ensure the accuracy and reproducibility of treatment performance. To do so, both DNA and RNA are extracted from the same biologic sample. Some existing protocols achieve simultaneous DNA and RNA extraction by splitting the lysate into two portions, then treating the portions of lysate with either DNase or RNase A. The drawback of this method, however, especially for precious samples, is that half of the DNA or RNA is lost.

This application note from Beckman Coulter Life Sciences demonstrates a simultaneous high-quality DNA and RNA extraction method for vector copy number and transgene expression testing that does not require splitting the sample lysate and shows consistent performance across species with 10 different tissue types and nine regions of the brain.

Quantitative real-time PCR (qPCR) is the most popular technique for quantifying nucleic acids. qPCR is simple to perform and offers excellent detection sensitivity, so scientists employ it in a diverse array of fields ranging from agricultural biology to molecular diagnostics. Like conventional PCR, qPCR uses denaturation, annealing, and extension cycles to exponentially produce additional copies of nucleic acid sequences of interest. Unlike conventional PCR, qPCR attaches a signal-emitting agent — typically a dye or a probe — to these sequences of interest. Signal magnitude after each cycle is measured and compared against controls to quantitate DNA abundance.

This guide from LGC Biosearch Technologies discusses considerations for selecting probes and primers for qPCR including sample type, research aims, and logistical obstacles, and it outlines probe options and how to troubleshoot probe-based qPCR.

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.

Different types of lipids affect cell response and viability. However, many lipid removal methods utilize solvents, such as Freon or chloroform, while other methods use solid phases that suffer from non-specific protein binding.

This report from Biotech Support Group highlights the ability of Cleanascite lipid removal reagent to clear lipid-associated matrix effects, including extracellular vesicles, which may influence cell response assays.

Tumor-associated macrophages (TAMs) located in the tumor microenvironment (TME), predominantly display an M2 protumor phenotype, and play a significant role in cancer cell survival and progression. Recent research suggests that the polarization of macrophages is orchestrated by fatty acid metabolism. Understanding the role of lipids in the TME has therefore become an important topic in tumor biology.

This case study from Biotech Support Group highlights the use of Cleanascite suspension reagent to remove artifacts in characterizing the influence of lipids and other factors bound to lipids on cell response in cancer.

The 10x Genomics Chromium single-cell platform has contributed to basic and translational research in immunology, developmental biology, and cancer by enabling the resolution of distinct cell populations within heterogeneous samples. Single-cell resolution of biological samples has accelerated our understanding of the complexity of living organisms and has opened up new possibilities for research in fields such as cancer research, genomics, and evolutionary biology.

Similarly, next-generation sequencing is an enabling technology for single-cell analysis, providing genomic information about the cell populations. Advancements in NGS over the last decade have contributed to faster, more accurate results while driving down the cost of experiments. The G4 Sequencing Platform combined with the 10x Genomics single-cell analysis platform allows researchers to achieve faster and more flexible sequencing while profiling tens of thousands of cells.

This application note from Singular Genomics demonstrates a workflow for producing data generated using 10x Chromium Single Cell 3’ Gene Expression assay with the novel G4 Sequencing Platform, demonstrating accuracy and technical reproducibility for single-cell analysis.

Single-cell RNA sequencing (scRNA-Seq) has revolutionized basic and translational research in immunology, developmental biology, and cancer by enabling the resolution of distinct cell populations within heterogeneous samples. However, there remains a need for cost-effective, high-throughput sequencing solutions to reduce the cost of scRNA-seq studies. We previously introduced the Max Read Kit, which enables higher output of short reads for the G4 Sequencing Platform, without a significant impact on read quality.

This poster from Singular Genomics evaluates the performance of the Max Read Kit for scRNA-seq by sequencing a 10x Genomics 3’ RNA-seq library prepared from human peripheral blood mononuclear cells (PBMC) using the G4, comparing results to those from the Illumina NextSeq 2000. We demonstrate high accuracy, reproducibility, and throughput with the Max Read sequencing kit.

Accurate and reproducible quantification of DNA is essential to achieving consistent NGS sequencing results. It is important to measure both the total quantity of DNA and the distribution of fragment sizes to achieve an optimal cluster density on the Illumina flow cell. Both factors impact data quality and total sequencing output. Among the common methods of DNA quantification, qPCR has the highest sensitivity.

However, qPCR setup is time intensive, and results are highly sensitive to small differences in pipetting. Automating NGS quantification provides a streamlined approach to getting reproducible, high-precision results while minimizing hands-on time.

This application note from Opentrons describes studies performed to evaluate two methods of qPCR normalization during the NGS workflow on the Opentrons OT-2 automated liquid handling platform, demonstrating the capabilities of the platform in automating NGS quantification and normalization in preparation for Illumina sequencing.

Next-generation sequencing has achieved widespread adoption as a tool for biological research and in vitro diagnostics. Despite this success, traditional NGS systems are limited by long analysis times, labor-intensive protocols, and the need for extensive sample batching to achieve cost-effective use. To address these limitations, Singular Genomics developed the G4 Platform for rapid and flexible sequencing.

This poster from Singular Genomics describes the application of the novel, higher-density F3 flow cell to perform 30x whole genome sequencing of the human reference cell line HG002 in a single flow cell on the G4 Sequencing Platform.