Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Analysis and Normalization of Real-Time PCR Experimental Data

(Protocol summary only for purposes of this preview site)

A real-time PCR experiment generates a large amount of raw numerical data, which are generally analyzed by basic software tools provided with the real-time PCR instrument. Because data analysis varies depending on the assay and instrument, it is necessary to refer to the manufacturer's instructions in order to analyze the data in an appropriate manner. However, even with data analysis software, it is good practice to examine the raw fluorescence data, and thereby evaluate the quality and reliability of the data, in order to generate reportable results. This involves three basic steps.

  • View the raw data (amplification plots), altering the baseline and threshold if necessary. CT values are a function of baseline and threshold values. Software default options provide some level of subjectivity, but these settings are not always appropriate and may need to be changed.
  • Verify the efficiency and sensitivity of the assay.
  • Apply a quantification method and normalize the data.


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Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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