Real-time PCR Data Export - ABI Instruments

1. Real-time PCR data: After Real-time PCR is completed, all data cannot be directly exported. Since each experiment is different, many default settings of the instrument will cause significant deviations from the final data result, so it is completed. After the PCR process, it is necessary to manually set the experimental results to extract scientific and effective experimental results, and then complete the analysis and judgment of the experimental results.

2. Data export adjustment steps: a adjust the reference fluorescence option; b adjust the baseline; c adjust the threshold

3. Concept:

a. Dye as the passive reference: the role of the reference fluorescence is to eliminate the optical path difference, adjust the linear shape of the amplification curve and the peak shape of the dissolution curve, related to the fluorescent dye mixture added by the PCR reaction, different company brands The mix contains different types of reference fluorescence, such as: Takara uses ROX, ABI has no reference fluorescence, and data extraction requires attention to the next reference fluorescence option, such as selection error, amplification curve or dissolution curve or will not Presented correctly.

b. Baseline: It means that the fluorescence signal does not change much during the first few cycles of the PCR amplification reaction. Close to a straight line, such a line is the baseline. The baseline adjustment includes the baseline starting point and the baseline focus. The starting point is generally set at 2-3 CTs. The ending point is set at the beginning of the amplification curve, that is, 2-3 CTs before the CT value entering the exponential growth period. The value, too late, will increase the CT value. If the default baseline period of the instrument is inconsistent with the above benchmark, it is likely that an artificial adjustment is required.

c. Fluorescence field value (threshold): The general instrument will use the fluorescence signal of the first 15 cycles (ie, the baseline period) of the PCR reaction as the fluorescence background signal, and the fluorescence field value defaults to 10 of the PCR standard deviation of 3-15 cycles of fluorescence signals. Times, the correct fluorescence domain value should be set in the exponential cycle number of PCR amplification.

4. StepOne reference fluorescence: Select and set the reference fluorescence in the plate setup

5. StepOne Baseline: Select the Analysis Setting button and the following dialog box appears.

• Check the Advanced Setting option to adjust each of the large Baseline End values ​​to the correct value.

• Finally click on the Apply Analysis Setting button

• The left graph adjusts the front baseline slightly upturned, and the baseline is adjusted after the map is adjusted.

• 6. StepOne threshold (Threshold): Select all the values ​​in the orifice plate. The following interface selects the log and target dialog boxes.

• Ticks the Tredshold Auto for each gene and the threshold value adjustment range is in the exponential growth phase.

The threshold value adjustment range principle is in the exponential growth period, in which the slope of the curve is the same and the ΔCT value is unchanged.

• Note that the same threshold results for all internal reference gene thresholds should be consistent, such ΔCT is comparable.