Applied Biosystems 7500 - Page 97

Chapter 5 Analyze the Experiment
View the Standard Curve
73
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
Notes
Analysis
Guidelines
When you analyze your own standard curve experiment, look for:
• Slope/amplification efficiency values – The amplification efficiency is calculated
using the slope of the regression line in the standard curve. A slope close to −3.3
indicates optimal, 100% PCR amplification efficiency. Factors that affect
amplification efficiency are:
– The range of standard quantities – For more accurate and precise efficiency
measurements, use a broad range (10
5
to 10
6
fold) of standard quantities.
– The number of standard replicates – For more accurate efficiency measurements,
include replicates to decrease the effects of pipetting inaccuracies.
– PCR inhibitors – PCR inhibitors in the reaction can reduce amplification
efficiency.
• R
2
values (correlation coefficient) – The R
2
value is a measure of the closeness of
fit between the regression line and the individual C
T
data points of the standard
reactions. A value of 1.00 indicates a perfect fit between the regression line and the
data points. An R
2
value >0.99 is desirable.
• C
T
values – The threshold cycle (C
T
) is the PCR cycle number at which the
fluorescence level meets the threshold. A C
T
value >8 and <35 is desirable. A C
T
value <8 indicates that there is too much template in the reaction. A C
T
value >35
indicates a low amount of target in the reaction; for C
T
values >35, expect a higher
standard deviation.
If your experiment does not meet the guidelines above, troubleshoot as follows:
• Omit wells (see “Omit Wells from the Analysis” on page 90).
or
• Rerun the experiment.
For More
Information
For more information on:
• The Standard Curve screen – Open the 7500 Software Help by clicking or
pressing F1.
• Amplification efficiency – Refer to the Amplification Efficiency of TaqMan
®
Gene
Expression Assays Application Note.