Published: 2011 ISBN: 978-1-904455-72-1
Price: GB £159 or US $310
An essential book for all scientists using PCR, real-time PCR, qPCR and related techniques. read more ...
Full details of this book at PCR Troubleshooting and Optimization. More figures at PCR Figures.
Chapter 2. Figure 1. A.) Cy0 method for determining quantitative differences between amplification curves (the point of inflection on each singular amplification plot is not fixed). This method does not assume equal efficiency for all reactions. B.) Graph A rotated 90 degrees showing the relationship between Cq vs. LOG of dRn (ROX-normalized reporter fluorescence in this case). C.) General approach to fluorescence-based estimation of efficiency of single amplification curves. Evaluation of sample amplification efficiencies are determined by examining a window of amplification FCq and relative fluorescence units (RFU) values between Fo and Fmax. Sigmoidal curve-fitting methods are based on this approach. This method does not assume equal efficiency for all reactions when curves are evaluated on a singular basis. D.) Threshold-based Cq method of amplification analysis wherein efficiencies are determined from the slope of target standard curves (the most prevalent method). The quantification cycle (Cq) for each reaction is determined by the point of intersection of each amplification plot with the same threshold line drawn through the most LOG-linear region of all same-target amplifications. Theoretically, at Cq, the same number of amplicons (Xn) has been generated by every sample. This method assumes equal efficiency for all reactions.
Further reading at PCR Troubleshooting and Optimization. More figures at PCR Figures.