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Overview

Applied Biosystems QuantStudio real-time PCR systems can detect changes as low as 1.5-fold. Most systems can also provide reproducible separation over 10 logs of input template.

 

Software provides an online toolkit for the analysis of the QuantStudio systems’ data. The software takes advantage of Connect, our cloud-based platform, to provide highly versatile analysis tools that are flexible, fast, easy to use and facilitate functional understanding of qPCR and related data.

 

These modules allow users to combine over 100 genotyping, gene expression, or other qPCR experiments into a project and analyze the data within minutes. Featured modules include Design and Analysis, High Resolution Melt (HRM) Analysis, Presence/Absence Analysis, Standard Curve, Genotyping, hPSC Scorecard Analysis, and Relative Quantification. See examples below.

Sensitivity

Detection as low as 1.5-fold discrimination

Distinct clusters indicating 2-fold and 1.5-fold discrimination on QuantStudio 7 Pro system with 99.9% confidence, equivalent to other Applied Biosystems platforms, QuantStudio 7 Flex and 7900HT instruments. Total liver cDNA samples with 2-fold and 1.5-fold differences in input were assessed. Runs were analyzed using their respective platform software, with auto threshold and baseline, then exported and analyzed using SAS JMP v13 statistical software.

The amplification plot shows detection of 1.5-fold changes in expression using the  QuantStudio 7 Pro system. A dilution series of the KAZ plasmid was amplified. The vertical axis is the change in fluorescence normalized to the ROX dye. The horizontal axis is increasing cycle number from left to right. This is verified by plotting as a standard curve. The vertical axis of the standard curve is the cycle threshold value and the horizontal axis is the dilution series increasing in concentration from left to right.

Amplification of KAZ plasmid DNA in 10-fold dilutions was performed using the 96-well block and shows highly reproducible separation over the 10 logs of input template amount. The vertical axis is the log of the background-subtracted fluorescence normalized to the ROX dye. The horizontal axis is increasing cycle number from left to right.

Single copy detection

Single copy of the KAZ plasmid in blue compared to NTC in purple.  The QuantStudio 7 Pro system consistently detects a single copy, clearly distinguishable from NTCs (43/48 positive wells). 

Multiplexing

Multiplex reaction detects 18S rRNA labeled with the VIC dye and TGFb gene with FAM-labeled TaqMan probes and primers. The reaction used the fast master mix and was run in 96 replicates. The vertical axis reflects the change in florescence normalized to the ROX dye, and the horizontal axis reflects increasing cycles from left to right. The CT values show a clear, consistent separation of products with a low standard deviation among replicates.

Genotyping

These plots demonstrate how improved visuals and allelic discrimination plots in the Genotyping app accurately reflect the true signals versus background noise.

HRM

The High-Resolution Melt (HRM) Analysis app is designed for post-PCR analysis to identify variation in nucleic acid sequences. The method detects small differences in PCR melting (dissociation) curves enabled by high-brightness, dsDNA-binding dyes in conjunction with QuantStudio systems. The system’s precise temperature ramp control, advanced data capture capabilities, and HRM analysis software makes it well suited for HRM analysis.

 

 

For Research Use Only. Not for use in diagnostic procedures.

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