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Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
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Quantification of siRNA using competitive qPCR.

Wei-li Liu1, Mark Stevenson, Leonard W Seymour

  • 1Department of Clinical Pharmacology, University of Oxford, Headington, Oxford, UK.

Nucleic Acids Research
|November 14, 2008
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Summary

We developed a novel PCR method to quantify short interfering RNA (siRNA) without RNA conversion. This competitive quantitative PCR (cqPCR) accurately measures siRNA delivery and stability, aiding therapeutic development.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Pharmacology

Background:

  • Short interfering RNA (siRNA) therapeutics require accurate quantification for efficacy.
  • Current methods for siRNA quantification often involve complex RNA conversion steps.
  • Assessing siRNA stability and cellular uptake is crucial for drug development.

Purpose of the Study:

  • To develop a direct, PCR-based method for quantifying siRNA.
  • To validate the method's ability to assess siRNA sequence specificity and stability.
  • To demonstrate the method's utility in quantifying siRNA delivery in cells.

Main Methods:

  • Developed a competitive quantitative PCR (cqPCR) assay using homologous DNA primers.
  • Designed primers and probes for amplifying HPV E6 or EGFP gene regions.
  • Tested siRNA's ability to compete with primers for template DNA binding.
  • Assessed the impact of siRNA modifications and degradation on amplification inhibition.
  • Quantified siRNA uptake in C33-A cells following delivery with different agents.

Main Results:

  • The cqPCR method showed a dose-dependent, linear increase in Ct values with increasing siRNA concentration.
  • 2'-O-methyl ribose-modified siRNA maintained inhibitory activity in serum, unlike unmodified siRNA.
  • Mismatch-bearing or truncated siRNAs failed to inhibit amplification, confirming sequence specificity.
  • cqPCR successfully quantified dose-dependent siRNA uptake in C33-A cells.
  • The method demonstrated discrimination between degraded and non-degraded siRNA sequences.

Conclusions:

  • The developed cqPCR assay provides a direct and accurate method for siRNA quantification.
  • This technique facilitates the assessment of siRNA stability and sequence-specific activity.
  • cqPCR enables reliable measurement of siRNA delivery and uptake in cellular models.
  • The method holds promise for determining siRNA pharmacokinetics and advancing therapeutic delivery strategies.