7900HT Fast Real-Time PCR System with 384-Well Block Module and Automation Accessory

The threshold is the numerical value assigned for each run that reflects the average dRn (change in fluorescence) during the initial cycles of PCR (baseline). The threshold is set by determining a statistically significant point (or value) above the baseline. You can manually adjust the threshold should you desire, to the place in the geometric phase where your replicates are tightest. For more information on how to manually set a threshold, please refer to the tutorial entitled Data Analysis on the ABI PRISM 7700: Setting Baselines and Thresholds (P/N 4370923). While this tutorial is based on the ABI PRISM 7700 system, the concepts are still valid for the Applied Biosystems real-time PCR instruments. You can search the document on our website by using the part number above as the keyword.

If you choose not to manually adjust the baselines and thresholds, the SDS software on the Applied Biosystems real-time PCR instruments has an Auto Ct algorithm that can be used to automatically generate baseline and threshold values for individual detectors. The algorithm calculates baseline and threshold parameters for a detector based on the assumption that the data exhibits the "typical" amplification curve. Experimental error (i.e. contamination, pipetting inaccuracies) can produce amplification curves that deviate significantly from a typical amplification curve. The data from these irregularities can affect the Auto Ct algorithm by causing it to generate incorrect baseline and threshold parameters for the associated detector. Therefore, Applied Biosystems recommends that after analysis of experimental data, you review all baseline and threshold parameters determined by the Auto Ct algorithm.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

It is possible to import a standard curve into the Design & Analysis Software (v2.x) if you are analyzing results from one of the following Applied Biosystems real-time PCR instruments:
• 7900HT Fast Real-Time PCR System
• 7500/7500 Fast Real-Time PCR System
• StepOne/StepOne Plus Real-Time PCR System
• ViiA 7 Real Time PCR System
• QuantStudio 1 Real-Time PCR System
• QuantStudio 3 & 5 Real-Time PCR System
• QuantStudio 6 & 7 Flex Real-Time PCR System
• QuantStudio 6 & 7 Pro Real-Time PCR Systems
• QuantStudio 12K Flex Real-Time PCR system

Follow the instructions below to import a standard curve for analysis in the Design & Analysis Software (v2.x):
1. Open the data file in the Design & Analysis Software (v2.x).
2. Click on Actions.
3. Click on "Standard Curve Analysis Setting".
4. Select "External Standard Curves" and click on "Import".
5. Browse to the proper .csv file and click "Open".
6. Click "Apply".

There are two main differences between a Fast 96-well and standard 96-well block for the Applied Biosystems real-time PCR systems:
• Reaction well volume: The Fast 96-well blocks have 0.1 mL reaction wells and are compatible with 0.1 mL plates and tubes. The standard 96-well blocks have 0.2 mL reaction wells and are compatible with 0.2 mL plates and tubes.
• Ramp rate: The fast 96-well blocks have a higher maximum ramp rate than standard blocks for the same real-time PCR system. However, both the fast 96-well and standard 96-well blocks are capable of running standard and fast chemistry.

Note: For newer instruments (QuantStudio 3 and 5 Real-Time PCR Systems and later) "0.1 mL" and "0.2 mL" are being used in place of "fast" and "standard" as designations for the two 96-well block formats.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

All our real-time PCR instruments are compatible with Thermo Fisher Cloud Apps except the 7500 Fast instrument where the eds files alone are compatible with Thermo Fisher Cloud Apps.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.