PCR Troubleshooting and Optimization Figure
Published: 2011 ISBN: 978-1-904455-72-1
Price: GB £159 or US 0
An essential book for all scientists using PCR, real-time PCR, qPCR and related techniques. read more ...
Figure from: PCR Troubleshooting and Optimization
Chapter 7. Figure 4. Calculation workflow. Identical relative quantities (RQ) of a given target between two samples (A and B) do not necessarily imply equal expression levels of the measured target by the applied assay. Differences in final cDNA concentration (represented by a different hue) or technical run-to-run variation (represented by a variable width) may obscure the picture of true differential expression. The normalization procedure removes the variation resulting from the use of different cDNA concentrations, thus yielding normalized relative quantities (NRQ) in which the expression level is only affected by possible run-to-run variation. This remaining variation can be removed by calibrating NRQs into calibrated normalized relative quantities (CNRQ) that truly reflect the differences in gene expression between the two samples. From a mathematical perspective normalization and inter-run calibration are equivalent procedures and are effected by division by a normalization factor (NF, geometric mean of reference gene NRQ values) or a calibration factor (CF, geometric mean of IRC sample NRQ values), respectively.
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