drug screening using peripheral blood mononuclear cells (pbmcs) and cell lysate preparation for mrna sequencing

Transcriptome Profiling for High-Throughput Drug Screening

The development of new drugs is a complex and time-consuming process that involves identifying potential drugs and their targets, optimizing and synthesizing drug candidates, and testing their efficacy and safety in preclinical and clinical trials1. Traditional methods for drug screening, i.e., the systematic assessment of libraries of candidate compounds for therapeutic purposes, involve the use of animal models or cell-based assays to test the effects on specific targets or pathways. While these methods have been successful in identifying drug candidates, they often did not provide sufficient insights into the complex molecular mechanisms underlying drug efficacy and also toxicity and mechanisms of potential side effects.
Assessing genome-wide transcriptional states presents a powerful approach to overcome current limitations in drug screening, as it enables comprehensive assessments of gene expression in response to drug treatments2. By measuring RNA transcripts in a genome-wide fashion expressed at a given time, transcriptomics aims to provide a holistic view of the transcriptional changes that occur in response to drugs, including changes in gene expression patterns, alternative splicing, and non-coding RNA expression3. This information can be used to determine drug targets, predict drug efficacy and toxicity, and optimize drug dosing and treatment regimens.
One of the key benefits of combining transcriptomics with unbiased drug screening is the potential to identify new drug targets that have not been previously considered. Conventional drug screening approaches often focus on established target molecules or pathways, hindering the identification of new targets and potentially resulting in drugs with unforeseen side effects and restricted effectiveness. Transcriptomics can overcome these limitations by providing insights into the molecular changes that occur in response to drug treatment, uncovering potential targets or pathways that may not have been previously considered2.
In addition to the identification of new drug targets, transcriptomics can also be used to predict drug efficacy and toxicity. By analyzing the gene expression patterns associated with drug responses, biomarkers can be developed that can be used to predict a patient's response to a particular drug or treatment regimen. This can also help to optimize drug dosing and reduce the risk of adverse side effects4.
Despite its potential benefits, the cost of transcriptomics remains a significant barrier to its widespread application in drug screening. Transcriptomic analysis requires specialized equipment, technical expertise, and data analysis, which can make it challenging for smaller research teams or organizations with limited funding to utilize transcriptomics in drug screening. However, the cost of transcriptomics has been steadily decreasing, making it more accessible to the research communities. Additionally, advancements in technology and data analysis methods have made transcriptomics more efficient and cost-effective, further increasing its accessibility2.
In this protocol, we describe a high-dimensional and explorative system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics analysis5,6. With this protocol, it is possible to reduce the cost per sample to 1/6th of the current cost of commercial solutions for full-length mRNA sequencing. The protocol requires only standard laboratory equipment, the only exception being the use of short-read sequencing technologies, which can be outsourced if sequencing instruments are not available in-house. The optimized mini-bulk protocol provides information-rich biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules.
The aim of the experiment is to screen for drug activity on PBMCs in different biological contexts. This protocol can be applied to any biological question where several drugs should be tested with a transcriptomic readout, giving a transcriptome-wide view of the cellular effect of the treatment.

Author Spotlight: Cost-Effective Transcriptomic Drug Screening - Unlocking New Targets 

Cost-Efficient Transcriptomic-Based Drug Screening

Transcriptomics enable us to gain a deep understanding of cellular program and their reaction towards external changes. However, despite a considerable reduction in library production and sequencing costs over the last decades, the application of this technology, at the scale needed for drug screening, is still unaffordable, hindering the great potential of these methods. Our study present a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics.The optimized mini-bulk protocol provides informative biological signal at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. One of the key benefits of combining transcriptomics with unbiased drug screening is the potential to identify new drug targets that have not been previously considered. Conventional drug screening approaches often focus on established target molecules or pathways, hindering the identification of new targets, and potentially resulting in drugs with unforeseen side effects and restricted effectiveness.

Watch this Scientific Journal Video about Drug Screening Using Peripheral Blood Mononuclear Cells PBMCs and Cell Lysate Preparation for mRNA Sequencing at JoVE.com

Drug Screening Using Peripheral Blood Mononuclear Cells (PBMCs) and Cell Lysate Preparation for mRNA Sequencing  

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109 Views.  German Center for Neurodegenerative Diseases (DZNE) eV.. Drug Screening Using Peripheral Blood Mononuclear Cells (PBMCs) and Cell Lysate Preparation for mRNA Sequencing

Video: Drug Screening Using Peripheral Blood Mononuclear Cells (PBMCs) and Cell Lysate Preparation for mRNA Sequencing  

cost-efficient transcriptomic-based drug screening

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

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cdna library preparation from the drug treated-peripheral blood mononuclear cell (pbmc) lysate for mrna sequencing

CDNA Library Preparation from the Drug Treated-Peripheral Blood Mononuclear Cell (PBMC) Lysate for mRNA Sequencing  

cDNA Library Preparation from the Drug Treated-Peripheral Blood Mononuclear Cell (PBMC) Lysate for mRNA Sequencing

whole blood purification and peripheral blood mononuclear cells (pbmcs) isolation using manual method

Whole Blood Purification and Peripheral Blood Mononuclear Cells (PBMCs) Isolation Using Manual Method

isolating human peripheral blood mononuclear cells from buffy coats via high throughput immunomagnetic bead separation

Author Spotlight: Advancements in PBMC Isolation &amp;#8211; Enhancing Throughput and Consistency in Research

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High-Throughput PBMC Isolation Using Immunomagnetic Beads

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peripheral blood mononuclear cells (pbmcs) purification using a high throughput automated bead-based method

Peripheral Blood Mononuclear Cells (PBMCs) Purification Using a High Throughput Automated Bead-Based Method

isolation of peripheral blood mononuclear cells (pbmcs) from human blood and differentiation of pbmcs into macrophages for phagocytosis assay

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay

preparation of peripheral blood mononuclear cell pellets and plasma from a single blood draw at clinical trial sites for biomarker analysis

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

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Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

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cryopreservation and bioenergetic evaluation of human peripheral blood mononuclear cells

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place...

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

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viability assay of trichoderma stromaticum conidia inside human peripheral blood mononuclear-derived macrophages

Author Spotlight: Unraveling the Interplay Between &lt;em&gt;Trichoderma stromaticum&lt;/em&gt; and the Mammalian Immune System

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Fungal Clearance Efficiency of Macrophages

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sequencing of mrna from whole blood using nanopore sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such...

isolation and cryopreservation of highly viable human peripheral blood mononuclear cells from whole blood: a guide for beginners

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

flotation-based t cell isolation, activation, and expansion from human peripheral blood mononuclear cell samples using microbubbles

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

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isolation of peripheral blood mononuclear cells from human buffy coats

In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further...

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isolating human peripheral blood mononuclear cells and cd4+ t cells from s&#233;zary syndrome patients for transcriptomic profiling

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&amp;#233;zary Syndrome Patients for Transcriptomic Profiling

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

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Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

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a flow cytometry assay to identify immune cell subsets in peripheral blood mononuclear cells

This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating...

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

isolation and cryopreservation of human peripheral blood mononuclear cells for bioenergetic studies

Isolation and Cryopreservation of Human Peripheral Blood Mononuclear Cells for Bioenergetic Studies  

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease ...