Name: Drug Screening Using Peripheral Blood Mononuclear Cells (PBMCs) and Cell Lysate Preparation for mRNA Sequencing
Uploaded: 2024-02-23T13:30:00
Description: 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

التنميط الترانسكريبتوم لفحص الأدوية عالية الإنتاجية

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.

تسليط الضوء على المؤلف: فحص الأدوية النسخي الفعال من حيث التكلفة - فتح أهداف جديدة 

فحص الأدوية القائم على النسخ الفعال من حيث التكلفة

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  

فحص الأدوية باستخدام خلايا الدم المحيطية أحادية النواة (PBMCs) وإعداد تحلل الخلايا لتسلسل mRNA

للبدء ، قم بإذابة قارورة التبريد الخاصة ب PBMC في حمام مائي عند 37 درجة مئوية لمدة دقيقتين إلى ثلاث دقائق مع قلبها برفق. ثم نقل الخلايا المذابة إلى أنبوب مخروطي 50 ملليلتر. اشطف قارورة التبريد بمليلتر واحد من وسط النمو الكامل الدافئ ، وأضف المحلول بالتنقيط إلى الخلايا الموجودة في الأنبوب المخروطي لأداء خطوة التخفيف الأولى.الآن الطرد المركزي تعليق الخلية لمدة خمس دقائق وقلب الأنبوب المخروطي بطلاقة لإزالة المادة الطافية. أعد تعليق الخلايا في ثلاثة ملليلتر من وسط النمو الكامل الدافئ. لعد الخلايا ، امزج 10 ميكرولتر من تعليق الخلية مع تريبان الأزرق.عد الخلايا باستخدام 10 ميكرولترات من محلول الخلايا الملطخة ، إما باستخدام عداد خلية آلي أو حجرة خلوية. استخدم وسط النمو الكامل الدافئ لتخفيف الخلايا لتحقيق تركيز نهائي قدره واحد في 10 أس ست خلايا لكل ملليلتر. ثم زرع 100 ميكرولتر من تعليق الخلية في كل بئر من صفيحة زراعة الخلايا المكونة من 96 بئرا.أضف 100 ميكرولتر من تخفيف الدواء مرتين إلى كل بئر. لحصاد الخلايا ، قم بطرد مركزي لوحة ثقافة الخلية لمدة 10 دقائق. استخدم مضخة تفريغ لإزالة المادة الطافية أثناء وضع طرف المضخة في الزاوية السفلية من البئر للاحتفاظ بالخلايا.أضف الآن 200 ميكرولتر من الثلج البارد PBS إلى كل بئر. بعد الطرد المركزي للوحة لمدة خمس دقائق ، قم بإزالة المادة الطافية. ثم أضف 15 ميكرولترا من محلول التحلل إلى كل بئر.باستخدام فيلم مانع للتسرب لاصق ، أغلق اللوحة للحماية من التلوث. دوامة اللوحة وأجهزة الطرد المركزي لمدة دقيقة واحدة. أخيرا ، اجمع ستة ميكرولترات من محلول الخلية المحللة في لوحة PCR.

Research

JoVE Journal

Biology

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

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 ...