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Masterclass_Leiden_2017_bonus_exercises


Masterclass: Bonus exercises.
This Zip file contains two Excel files with example data and 2 pdf files with the manuals for two exercises about removal of between-plate variation. The first pdf file contains the instructions on how to remove the between-run variation for a PCR experiment with 6 plates that have already been analysed with LinRegPCR and annotated. The 7th sheet in this Excel file contains a pair of pre-prepared graphs that illustrate the effect of Factor-qPCR. The second pdf file gives the short instructions on how to analyse two PCR runs with LinRegPCR, remove the between-plate variation, perform reference gene normalisation and then draw a graph of the results per operator. The instructions are only stepwise, when needed you can refer to earlier exercises for deatils on how to perform ach step. The last part, normalisation and graphs, is very similar to exercise 2B and can be skipped.


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References

  • 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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