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

Rocco Sciarrillo; Anna Wojtuszkiewicz; Irsan E. Kooi; Valentina E. G&#243;mez; Ugo Boggi; Gerrit Jansen; Gert-Jan Kaspers; Jacqueline Cloos; Elisa Giovannetti

doi:10.3791/54714

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

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Cancer Research

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JoVE Journal Cancer Research

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

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09:58 min

December 9, 2016

DOI: 10.3791/54714-v

Rocco Sciarrillo^1,2,3, Anna Wojtuszkiewicz¹, Irsan E. Kooi⁴, Valentina E. Gómez³, Ugo Boggi⁵, Gerrit Jansen⁶, Gert-Jan Kaspers^1,7, Jacqueline Cloos*¹, Elisa Giovannetti*^3,8,9

¹Department of Pediatric Oncology/Hematology,VU University Medical Center, ²Department of Hematology,VU University Medical Center, ³Department of Medical Oncology,VU University Medical Center, ⁴Department of Clinical Genetics,VU University Medical Center, ⁵Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana,Universita’ di Pisa, ⁶Amsterdam Immunology and Rheumatology Center,VU University Medical Center, ⁷Princess Máxima Center for Pediatric Oncology, ⁸Cancer Pharmacology Lab, AIRC Start-Up Unit,University of Pisa, ⁹Institute of Nanoscience and Nanotechnology,CNR-Nano

Overview

This protocol investigates the association of aberrant splicing with drug resistance in solid tumors and hematological malignancies. By analyzing transcriptomic profiles through RNA-seq, it aims to enhance understanding of chemotherapy resistance mechanisms.

Key Study Components

Area of Science

Molecular pharmacology
Chemotherapy resistance
Transcriptomic analysis

Background

Aberrant splicing can influence drug resistance.
Understanding post-transcriptional mechanisms is crucial for improving treatment outcomes.
This study combines cytotoxicity screening with next-generation sequencing.
Insights can be applied to various systems, including patient samples.

Purpose of Study

To investigate mechanisms of drug resistance at the molecular level.
To validate candidate genes associated with resistance.
To improve early detection of drug resistance.

Methods Used

RNA sequencing (RNA-seq) for transcriptomic profiling.
Quantitative reverse transcription PCR (qRT-PCR) for gene validation.
Cytotoxicity screening protocols.
Analysis of in vitro models of solid tumors and hematological malignancies.

Main Results

Identification of key genes involved in drug resistance.
Establishment of a reliable method for validating transcriptomic data.
Insights into genome-wide determinants of drug resistance.
Potential applications to primary patient samples and xenografts.

Conclusions

This protocol enhances understanding of chemotherapy failure mechanisms.
It provides a framework for future research on drug resistance.
Combining techniques can lead to improved therapeutic strategies.

Frequently Asked Questions

What is the main focus of this study?

The study focuses on the impact of aberrant splicing on drug resistance in tumors.

How does this protocol improve drug resistance detection?

It combines transcriptomic analysis with cytotoxicity screening for better insights.

Can this method be applied to patient samples?

Yes, it can be applied to primary patient samples and xenografts.

What techniques are used in this protocol?

RNA-seq and qRT-PCR are the primary techniques used.

What are the expected outcomes of this research?

The research aims to identify mechanisms of drug resistance and improve treatment strategies.

Why is understanding aberrant splicing important?

Aberrant splicing can significantly affect drug resistance profiles in cancer treatment.

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 genes.

The overall goal of this protocol is to investigate the association of aberrant splicing with drug resistance profiles in in vitro models of solid tumors and hematological malignancies. This method can help answer key question in the field of molecular pharmacology and chemotherapy resistance such as which are the mechanism underlying drug resistance on post-transcriptional level? The main advantage of this technique is that it combines well established cytotoxicity screening protocol with powerful next generation sequencing based transcriptomic analysis.

This technique could lead to a better understanding of the molecular mechanisms underlying chemotherapy failure and it will improve the early detection of drug resistance. This method can provide insight into genome-wide determinants of drug resistance in cell line models. But it can also be applied to other systems such as primary patient samples and xenograft.

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