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qPCR

qPCR. Taylor Britton. Introduction. Quantitative polymerase chain reaction ( qPCR ) Monitor the amplification of a targeted DNA molecule during PCR in real-time. Limitations of PCR quantification. Poor precision Low sensitivity.

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qPCR

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  1. qPCR Taylor Britton

  2. Introduction • Quantitative polymerase chain reaction (qPCR) • Monitor the amplification of a targeted DNA molecule during PCR in real-time

  3. Limitations of PCR quantification • Poor precision • Low sensitivity http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

  4. History of qPCR and RT-qPCR • Higuchi, Fockler, Dollinger, and Watson (1993) Higuchi, R., Fockler, C., Dollinger, G. & Watson, R. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat. Biotechnol.11, 1026–1030 (1993).

  5. Differences between RT-PCR, qPCR, and RT-qPCR • RT-PCR Reverse transcriptase PCR • qPCR Quantitative PCR (also referred to as real time PCR) • RT-qPCR Reverse transcriptase quantitative PCR

  6. How to make cDNA http://www.mun.ca/biology/desmid/brian/BIOL4900/CB18_31.html

  7. How does it work • Fluorescent signal • CT (cycle threshold) • Two methods dsDNA-Binding Dyes Taqman probes (fluorogneic probe) • Absolute vs relative quantitation Virus DNA/RNA Extraction Kit II VR050/VR100/VR300 | Geneaid

  8. Relative Quantification • Calibrators • Internal Control (house keeping/reference genes) https://www.gene-quantification.de/qiagen-qpcr-sample-assay-tech-guide-2010.pdf Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods25, 402–408 (2001)

  9. dsDNA-Binding dyes Extension cont End of cycle Annealing Extension Quantitative PCR & Digital PCR Detection Methods - PCR Technologies Guide. Sigma-Aldrich

  10. Taqman probe Taqman probe qPCR Method. Available at: http://www.sinobiological.com/taqman-probe-qpcr-method-cro-service.html.

  11. Comparison of dsDNA-Binding dyes and Taqman probes

  12. What it looks like and Detection Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye | LSR | Bio-Rad.

  13. Pros and Applications • Sensitive, no gel, and no end point measurements • Determine copy number of gene • Quantification of gene expression • DNA damage • Drug Therapy • Quality control and assay validation • Pathogen detection during infection • Genotyping

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