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Related Concept Videos

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
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Measuring RNAi knockdown using qPCR.

Stuart Milstein1, Michael Nguyen, Rachel Meyers

  • 1Alnylam Pharmaceuticals, Cambridge, MA, USA.

Methods in Enzymology
|November 5, 2013
PubMed
Summary
This summary is machine-generated.

This study presents a scalable method for measuring gene expression changes after RNA interference (RNAi) in mammalian cells. The protocol helps identify effective small interfering RNAs (siRNAs) for gene knockdown and assess their potency.

Keywords:
Branched deoxyribonucleic acid assay (bDNA)Fluorescence resonance energy transfer (FRET)Quantitative PCR (qPCR)RNA isolation using magmax™ magnetic bead purificationRNAi knockdown measurementRibonucleic acid (RNA)TransfectioncDNA synthesis

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • RNA interference (RNAi) is a key tool for gene silencing.
  • Accurate measurement of gene expression changes is crucial for RNAi studies.
  • Identifying potent small interfering RNAs (siRNAs) requires efficient screening methods.

Purpose of the Study:

  • To describe a scalable method for measuring gene expression changes post-RNAi in mammalian cells.
  • To provide a protocol for identifying effective siRNAs and assessing their potency.
  • To enable efficient screening of siRNA panels targeting specific genes.

Main Methods:

  • Cell transfection and total RNA isolation.
  • Complementary DNA (cDNA) synthesis.
  • Quantitative Polymerase Chain Reaction (qPCR) using multiplexed TaqMan dual hydrolysis probes with relative quantification.

Main Results:

  • The described methods allow for the assessment of relative gene knockdown by various siRNAs.
  • The protocol facilitates the identification of the most effective siRNA molecules.
  • Dose-response screening can determine the potency of selected siRNAs.

Conclusions:

  • This scalable qPCR-based protocol provides a robust method for evaluating RNAi efficiency in mammalian cells.
  • The two-phase screening approach (single-dose followed by dose-response) optimizes the identification of potent siRNAs.
  • This method is valuable for both basic research and therapeutic development involving gene silencing.