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Masterclass: Analysis of qPCR amplification curves.
This Zip file contains an Excel file with example data files (masterclass_data_2017.xls) and 2 pdf files with exercises. The first pdf file gives instructions on how to install LinRegPCR and teaches about the basics of the program: import data into the program, handle deviating amplification curves, perform a baseline subtraction and save the results to Excel. The second pdf file focusses on effect of primer concentration on plateau and baseline levels and on analysis of gene expression data. For detailed information on the "how and why" of the LinRegPCR program you should refer to the manual and to papers that we wrote on the analysis of qPCR data. Each exercise will take about 1 hour to complete.

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  • Ramakers et al. Assumption-free analysis of quantitative real-time PCR data. Neurosci Letters 339: 62-66, 2003
  • Ruijter et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Research 37: e45, 2009
  • Tuomi et al. Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value. Methods 50: 313-322, 2010
  • Ruijter et al. Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications. Methods 59: 32-46, 2013
  • Ruijter et al. Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency. Microchimica Acta 2014
  • Ruijter JM, Ruiz Villalba A, Hellemans J, Untergasser A, van den Hoff MJB. Removal of between-run variation in a multi-plate qPCR experiment. Biomolecular Detection and Quantification 5:10-14, 2015.

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Version History

Workshop version 1.1: Released February 2014.

2016 version of the workshop.
The file contains:

  • masterclass_exercises_2017_1.pdf
  • masterclass_exercises_2017_2.pdf
  • masterclass_data_2017.xls

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Leiden_amplification_curves (automated content)