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Experimental RNAi

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...

RNA Interference

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...

RNA Interference

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...

siRNA - Small Interfering RNAs

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...

Rous Sarcoma Virus (RSV) and Cancer

Rous Sarcoma Virus (RSV) and Cancer

Rous Sarcoma virus or RSV was discovered by F. Peyton Rous in the year 1911 as a filterable transmissible agent that could cause tumors in chickens. He won a Nobel Prize for this discovery in 1966. His experiments clearly demonstrated that some cancers could be caused by infectious agents and led to the discovery of many more cancer-causing viruses in animals as well as humans.
RSV is a retrovirus that contains two copies of a plus-strand RNA genome. Its genome consists of four main open...

Rous Sarcoma Virus (RSV) and Cancer

Rous Sarcoma Virus (RSV) and Cancer

Rous Sarcoma virus or RSV was discovered by F. Peyton Rous in the year 1911 as a filterable transmissible agent that could cause tumors in chickens. He won a Nobel Prize for this discovery in 1966. His experiments clearly demonstrated that some cancers could be caused by infectious agents and led to the discovery of many more cancer-causing viruses in animals as well as humans.
RSV is a retrovirus that contains two copies of a plus-strand RNA genome. Its genome consists of four main open...

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Related Experiment Video

Updated: May 21, 2026

DNA Vector-based RNA Interference to Study Gene Function in Cancer

DNA Vector-based RNA Interference to Study Gene Function in Cancer

Published on: June 4, 2012

DNA vector-based RNA interference to study gene function in cancer.

Daniel B Stovall¹, Meimei Wan, Qiang Zhang

¹Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, USA.

Journal of Visualized Experiments : Jove

|June 20, 2012

Summary

This summary is machine-generated.

RNA interference (RNAi) is a powerful tool for gene silencing, offering advantages over genetic deletion for studying gene function. This study details a lentiviral vector-based RNAi method for gene silencing and its application in cancer research.

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DNA Vector-based RNA Interference to Study Gene Function in Cancer

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RNAi Interference by dsRNA Injection into Drosophila Embryos

RNAi Interference by dsRNA Injection into Drosophila Embryos

Published on: April 11, 2011

Area of Science:

Molecular Biology
Gene Expression Regulation
Cancer Research

Background:

RNA interference (RNAi) inhibits gene expression by degrading target messenger RNAs (mRNAs).
RNAi technology, particularly small hairpin RNA (shRNA) expressed from DNA vectors, enables long-term and inducible gene silencing.
Lentiviral vectors enhance shRNA delivery and stable expression for functional studies.

Purpose of the Study:

To describe a detailed procedure for DNA vector-based RNAi using lentiviral vectors for gene function determination.
To demonstrate the efficacy of an inducible shRNA system for silencing essential genes in cancer research.
To evaluate the impact of gene silencing on tumor formation using a mouse xenograft model.

Main Methods:

Construction of lentiviral vectors expressing shRNA via PCR amplification and 3-fragment ligation.
Production of lentivirus using calcium phosphate precipitation for efficient transfection of 293T cells.
Application of a Tet-On inducible shRNA system to silence Yin Yang 1 (YY1) in breast cancer cells and assessment in a mouse xenograft model.

Main Results:

An efficient method for generating shRNA-containing lentiviral constructs was established, allowing easy transfer of expression cassettes.
Calcium phosphate precipitation achieved over 90% transfection efficiency for lentivirus production.
The Tet-On inducible shRNA system successfully silenced the potential oncogene YY1, demonstrating its effect on tumor formation.

Conclusions:

DNA vector-based RNAi technology, particularly with lentiviral delivery, provides a robust method for gene function studies.
Inducible shRNA systems are valuable for investigating genes essential for cell proliferation and tumor development.
This protocol facilitates efficient gene silencing and functional analysis in cancer research models.