mrna sequencing to understand the cellular effect of drug treatment

Profilage du transcriptome pour le criblage de médicaments à haut débit

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.

Pleins feux sur l’auteur : Dépistage transcriptomique rentable des médicaments - Débloquer de nouvelles cibles 

Dépistage de drogues basé sur la transcriptomique rentable

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  

Séquençage de l’ARNm pour comprendre l’effet cellulaire d’un traitement médicamenteux

Pour commencer, combinez des bibliothèques indexées de manière unique dans un rapport équimolaire. Mesurez la concentration de la piscine finale à l’aide d’un test à haute sensibilité pour calculer la molarité de l’échantillon à l’aide de cette équation. Maintenant, chargez la cellule d’écoulement en suivant les spécifications de l’instrument et toute optimisation expérimentale.Sélectionnez Séquence à l’écran pour commencer la configuration de l’exécution. Suivez les instructions à l’écran pour charger la séquence de la cellule d’écoulement par cartouche de synthèse, cartouche de clustering et cartouche tampon. Une fois tous les réactifs reconnus par l’équipement, ajoutez le nom de l’exécution et la configuration de l’exécution.Spécifiez les détails de séquençage en tant que paire pour terminer, les deux lectures ayant une longueur de 51 paires de bases. De plus, séquencez deux lectures d’index de huit paires de bases chacune. Déterminez le répertoire de sortie pour le stockage des données et appuyez sur Vérifier.Après avoir vérifié l’exactitude de tous les paramètres de séquençage, sélectionnez Démarrer l’exécution pour commencer le séquençage.

mrna-sequencing-to-understand-the-cellular-effect-of-drug-treatment

Research

JoVE Journal

Biology

mRNA Sequencing to Understand the Cellular Effect of Drug Treatment

79 vues. Pour commencer, combinez des bibliothèques indexées de manière unique dans un rapport équimolaire. Mesurez la concentration de la piscine finale à l’aide d’un test à haute sensibilité pour calculer la molarité de l’échantillon à l’aide de cette équation. Maintenant, chargez la cellule d’écoulement en suivant les spécifications de l’instrument et toute optimisation expérimentale.Sélectionnez Séquence à l’écran pour commencer la configuration de l’exécu...

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

Cost-Efficient Transcriptomic-Based Drug Screening

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 ...

Recherche

Biologie

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

Criblage de médicaments à l’aide de cellules mononucléées du sang périphérique (PBMC) et préparation de lysats cellulaires pour le séquençage de l’ARNm

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  

Préparation d’une banque d’ADNc à partir du lysat de cellules mononucléées du sang périphérique (PBMC) traité par un médicament pour le séquençage de l’ARNm