mrna sequencing to understand the cellular effect of drug treatment

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 MRNA Sequencing to Understand the Cellular Effect of Drug Treatment at JoVE.com

MRNA Sequencing to Understand the Cellular Effect of Drug Treatment  

mRNA Sequencing to Understand the Cellular Effect of Drug Treatment

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71 Views.  German Center for Neurodegenerative Diseases (DZNE) eV.. mRNA Sequencing to Understand the Cellular Effect of Drug Treatment

Video: MRNA Sequencing to Understand the Cellular Effect of Drug Treatment  

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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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...

biotin-based pulldown assay to validate mrna targets of cellular mirnas

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain...

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

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

Author Spotlight: Decoding RNA Methylation&#039;s Role in Pancreatic Cancer - A Single-Base Resolution Study 

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of...

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

brain tumor stem cell (btsc) migration assay for drug treatment studies: an in vitro live-cell imaging technique to analyze the effect of drug treatment on migration of btscs

In this video, we perform brain tumor stem cell migration assay to analyze the effect of the drug of interest on the migration of live brain tumor stem cells using chemotaxis migration plate. Live-cell imaging facilitates the visualization and quantification of cell migration during the...

Brain Tumor Stem Cell (BTSC) Migration Assay for Drug Treatment Studies: An In Vitro Live-cell Imaging Technique to Analyze the Effect of Drug Treatment on Migration of BTSCs

At the end of the incubation, aspirate the collagen solution from each well without scratching the collagen coating and wash the wells two times with sterile PBS. After the last wash, remove the insert from the plate and add 225 microliters of growth factor-free media supplemented with 10% FBS to each bottom reservoir well of the chemotaxis...

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

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

multiplexed single cell mrna sequencing analysis of mouse embryonic cells

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch...

trans-tympanic drug delivery for the treatment of ototoxicity

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as...

brain tumor stem cell (btsc) neurosphere invasion assay: an in vitro assay to analyze the effect of drug treatment on btsc invasion from neurosphere

This video demonstrates the in vitro brain tumor stem cell (BTSC) invasion assay to determine the effect of drug treatment on the invasion capacity of BTSCs from the neurosphere via live-cell imaging...

Brain Tumor Stem Cell (BTSC) Neurosphere Invasion Assay: An In Vitro Assay to Analyze the Effect of Drug Treatment on BTSC Invasion from Neurosphere

Aspirate as much of the media from the bottom of each 1.5-milliliter tube as possible without losing the neurosphere pellet and gently resuspend the neurospheres in 500 microliters of freshly prepared 0.4-milligram per milliliter type I collagen solution. Transfer 100 microliters of the collagen suspended neurospheres to three wells per condition to a new 96-well plate on ice and allow the neurospheres to settle for 5...

using rna-sequencing to detect novel splice variants related to drug resistance in in vitro cancer models

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate...

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

analyzing the images of caenorhabditis elegans after drug treatment

Analyzing the Images of Caenorhabditis elegans after Drug Treatment  

Analyzing the Images of Caenorhabditis elegans after Drug Treatment

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is...

using the e1a minigene tool to study mrna splicing changes

Using the E1A Minigene Tool to Study mRNA Splicing Changes

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic...

models and methods to evaluate transport of drug delivery systems across cellular barriers

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body.  This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI)...

detecting the effect of dio on the mrna and protein levels of c1galt1 and cosmc in dakiki cells

Detecting the Effect of DIO on the mRNA and Protein Levels of C1GALT1 and Cosmc in DAKIKI Cells

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 ...