An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

Hyun  Ah Seo; Cho  Yean Hwang; Sokviseth Moeng; Jong Kook Park

doi:10.3791/59628

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tu...

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Genetics

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JoVE Journal Genetics

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

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09:45 min

May 21, 2019

DOI: 10.3791/59628-v

Hyun Ah Seo¹, Cho Yean Hwang¹, Sokviseth Moeng¹, Jong Kook Park¹

¹Department of Biomedical Science and Research Institute for Bioscience & Biotechnology,Hallym University

Overview

This protocol describes a method to identify microRNA targets and understand their functions, which is essential for studying biological processes in health and disease. It utilizes various assays to validate the direct targets of microRNAs, providing insights into their roles, particularly in cancer therapy.

Key Study Components

Area of Science

Neuroscience
MicroRNA research
Cancer biology

Background

MicroRNAs play a crucial role in regulating biological processes.
Understanding microRNA functions can aid in cancer therapy development.
Identifying microRNA targets is challenging but necessary for functional studies.
This protocol is designed to assist researchers in studying microRNAs.

Purpose of Study

To identify microRNA targets and validate their functions.
To provide insights into the implications of microRNAs in cancer therapy.
To establish a fundamental method for new researchers studying microRNAs.

Methods Used

Probe-based real-time polymerase chain reaction (PCR)
Sulforhodamine B (SRB) assay
3’ untranslated regions (3’ UTR) cloning
Luciferase assay

Main Results

Successful identification of microRNA targets.
Validation of microRNA functions in biological processes.
Insights into the role of microRNAs in cancer therapy.
Establishment of a reproducible protocol for future studies.

Conclusions

This protocol enhances understanding of microRNA functions.
It provides a foundation for further research in microRNA-target interactions.
The methods outlined are beneficial for evaluating anti-cancer drugs.

Frequently Asked Questions

What is the significance of microRNAs in biology?

MicroRNAs regulate various biological processes, influencing gene expression and cellular functions.

How does this protocol assist new researchers?

It provides a fundamental method to study microRNAs and their targets, facilitating understanding of their roles.

What assays are used in this protocol?

The protocol uses real-time PCR, SRB assay, 3' UTR cloning, and luciferase assays.

Can this protocol be applied to cancer research?

Yes, it specifically addresses the implications of microRNAs in cancer therapy.

What is the role of the half-maximal inhibitory concentration in this study?

It is used to evaluate the efficacy of microRNAs and other anti-cancer drugs.

How are microRNA targets validated?

Through various assays that confirm the interaction between microRNAs and their target genes.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3’ untranslated regions (3’ UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

To access neural microRNA target interactions with MicroRNA functions is critical to understand how MicroRNAs regulate various biological processes in both healthy and diseased states. This protocol describes the reproduce through strategy to identify microRNA targets and the functional microRNAs as the validation of direct target of microRNAs is challenging. Our protocol can provide insights on the function of individual microRNAs and the implication of a microRNA in cancer therapy.

Calculation of the half-maximal inhibitory concentration is an important method, not only for microRNA studies but for the efficacious evaluation of other anti-cancer drugs. Our protocol will be helpful to new researchers since we described the fundamental method to understand the level of microRNAs in a cell. To begin the protocol, count the cells with the cell counter.

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