abi 7500 - Page 108

Chapter 5 Analyze the Relative Standard Curve Experiment
View the Standard Curve
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
84
Notes
Analysis
Guidelines
When you analyze your own relative standard curve experiment, look for:
• Slope/amplification efficiency values – 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.
– 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) – 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 PCR cycle number at which the fluorescence level equals 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 105).
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.