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Published on: August 3, 2011
Validation of short interfering RNA knockdowns by quantitative real-time PCR
Sukru Tuzmen1, Jeff Kiefer, Spyro Mousses
1Pharmaceutical Genomics Division, Translational Genomics Institute, Scottsdale, AZ, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 3, 2007
Summary
Short interfering RNA (siRNA) enables sequence-specific gene silencing by exploiting RNA interference (RNAi). This study details methods for validating siRNA gene silencing effects, crucial for cancer research.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- RNA interference (RNAi) is a natural cellular mechanism for gene regulation.
- Double-stranded RNA is processed into short interfering RNA (siRNA) that degrades specific messenger RNA transcripts.
- Synthetic siRNA can be used to experimentally silence gene expression in mammalian cells.
Purpose of the Study:
- To present methods and procedures for validating siRNA-mediated gene silencing.
- To demonstrate the application of these methods using a Cancer Gene Library.
- To validate gene silencing for siRNAs that produce a phenotype in high-throughput screening.
Main Methods:
- Utilizing a Cancer Gene Library of 278 siRNAs targeting 139 oncogenes and tumor suppressor genes.
- Performing high-throughput RNAi phenotype analysis.
- Validating gene silencing using quantitative real-time PCR (qPCR) assays.
Main Results:
- Methods for analyzing sequence-specific gene silencing are presented.
- Validation of effective siRNAs is demonstrated through gene expression analysis.
- qPCR assays measure the extent of target gene silencing and its effects on gene expression endpoints.
Conclusions:
- The presented methods effectively validate siRNA-mediated gene silencing.
- Quantitative gene expression analysis is essential for confirming siRNA efficacy.
- This approach aids in identifying and validating therapeutic gene targets in cancer research.
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