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Related Experiment Videos

Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR.

D Klein1, M Denis, C Ricordi

  • 1Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.

Molecular Biotechnology
|July 13, 2000
PubMed
Summary

Reverse transcriptase real-time PCR (RT-qPCR) offers a standardized, low-contamination method for quantifying gene expression. This study utilized RT-qPCR to efficiently screen ribozymes targeting CD95 transcripts in mouse insulinoma cells.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Expression Analysis

Background:

  • Classical quantitative PCR methods present challenges in standardization and are prone to contamination.
  • Real-time PCR (PCR) offers a more streamlined and reliable approach for quantitative analysis of nucleic acids.

Purpose of the Study:

  • To evaluate the efficiency of anti-Fas ribozymes using quantitative analysis of CD95 (Fas) transcripts.
  • To establish and validate the reverse transcriptase real-time PCR (RT-qPCR) method for gene transcript quantification.

Main Methods:

  • Utilized reverse transcriptase real-time PCR (RT-qPCR) for quantifying CD95 (Fas) transcripts.
  • Employed melting-curve analysis to determine optimal fluorescent data collection temperature.
  • Constructed standard curves for accurate quantification of unknown sample copy numbers.

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  • Validated melting-curve profiles with gel electrophoresis for product size and melting temperature confirmation.

    • Main Results:

    • RT-qPCR provided a fast (under 1 hour, excluding RNA isolation and cDNA synthesis) and reproducible method.
    • Demonstrated a coefficient of variance of 15% within the 10^4-10^6 gene copy range.
    • Successfully quantified CD95 (Fas) transcripts to assess ribozyme cleavage efficiency.

    Conclusions:

    • RT-qPCR is a suitable technique for the high-throughput screening of ribozymes.
    • The method is highly efficient, standardized, and minimizes contamination risk.
    • Validated RT-qPCR offers a reliable approach for gene expression studies.