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Updated: Jul 6, 2026
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
A step-by-step procedure to analyze the efficacy of siRNA using real-time PCR
Angie Cheng1, Charles L Johnson, Lance P Ford
1Ambion, Applied Biosystems Inc., Austin, TX, USA.
Abstract:
Knockdown of cellular RNA using short interfering RNA has enabled researchers to perform loss-of-function (LOF) experiments in a wide variety of cell types and model systems. RNA interference techniques and reagents have made possible experiments that test everything from the analysis of function of single genes to screening for genes that are involved in critical biological pathways on a genome-wide scale. Although siRNA experiments are generally common practice in research laboratories, it is still important to keep in mind that many factors can influence efficacy of knockdown. A properly designed siRNA, optimized protocols of siRNA delivery, and an appropriate and well-optimized readout are all critical parameters for ensuring the success of your experiment. In this chapter, we provide step-by-step procedures for performing an siRNA knockdown experiment from cell culture to analysis of knockdown using quantitative real-time PCR.
Insights
Short interfering RNA (siRNA) enables loss-of-function studies for gene analysis and genome-wide screening. This guide details optimizing siRNA experiments, from cell culture to quantitative real-time PCR analysis, for successful knockdown.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Short interfering RNA (siRNA) is a key tool for RNA interference (RNAi) enabling gene knockdown.
- siRNA facilitates loss-of-function (LOF) studies across diverse cell types and model systems.
- Applications range from single-gene analysis to large-scale genomic screening of biological pathways.
Purpose of the Study:
- To provide a comprehensive, step-by-step protocol for conducting siRNA knockdown experiments.
- To highlight critical factors influencing siRNA efficacy, including design, delivery, and analysis.
- To guide researchers in optimizing their siRNA experiments for reliable results.
Main Methods:
- Detailed procedures for cell culture maintenance and preparation.
- Step-by-step guidance on siRNA design and delivery optimization.
- Methods for analyzing knockdown efficiency using quantitative real-time PCR (qRT-PCR).
Main Results:
- Successful implementation of siRNA knockdown protocols is demonstrated.
- Optimization strategies for siRNA design and delivery are presented.
- Validation of knockdown efficacy through qRT-PCR analysis is detailed.
Conclusions:
- Effective siRNA knockdown requires careful optimization of multiple experimental parameters.
- Standardized protocols ensure reproducibility and success in loss-of-function studies.
- This chapter serves as a practical resource for researchers performing siRNA experiments.
