TATAA Interplate Calibrator

Variation Compensation

The TATAA Interplate Calibrator (IPC) makes it possible for you to compensate for variations between qPCR runs. For practical reasons many qPCR studies involve the use of samples that are processed in more than a single batch or in which the sample set is extended over time. Even over a short time period, variation between qPCR processing runs is observed due to different baseline subtractions and threshold settings.


When to use TATAA Interplate Calibrator

The bias is NOT introduced when using the “all samples” or “all assays” plate layout or when performing ΔΔCq based analysis, but it is the “mixed” layout for which interplate calibration is needed (Figure 1). The TATAA IPC sample material is provided in ready-to-use aliquots and is a very stable template that is amplified with a highly robust assay. The TATAA IPC should be included in all qPCR runs. Any differences in the measured IPC Cq-values among the runs reflect the bias introduced by the instrument and can then compensated for.



Figure 1: Different options for the design of a multi-plate qPCR study for ΔΔCq based analysis. Top left: the study contains 24 samples and 16 genes and requires four runs with 96-well block instruments (y-axis: samples, x-axis: genes). Top right: “All samples” are always assayed on the same plate. Bottom left: “All genes” are always assayed on the same plate. Bottom right: “Mixed“ design which requires interplate calibration.



TATAA Interplate Calibrator is:

  • provided in aliquots (stored at -20°C) for easy and flexible use and long term stability
  • a robust assay that performs excellent in most mastermixes and over a wide range of annealing temperatures
  • a stable template at optimum concentration that produces Cq ≈ 15 -20 under most conditions

The Cq of the IPC is measured with high accuracy. The TATAA IPC has been extensively optimized to reliably and predictably amplify, providing highly reproducible Cq-values. However, it is still recommended as good practice to run technical replicates of the TATAA IPC (preferably at least triplicates, Figure 2). Poor assays should never be used as interplate calibrators, since the noise contributed by these measurements may in turn worsen the quality of the data rather than improving it. It is sufficient to run one set of IPC replicates for each instrument channel used within a study if a common threshold is set. Hence, for most singleplex assays, technical replicates of a single IPC are sufficient. It is not recommended to perform separate interplate calibrations for each assay, since the noise contributed by the independent corrections is likely to reduce data quality.



Figure 2: The relationship between the standard error of the mean (SEM) and the number of replicates used in runs with identical settings, except for them being processed on different instruments (see figure legend). LC480 shows two different threshold settings on one run.



Absolute Quantification

The TATAA IPC is also suited for absolute quantification. The recommended strategy is to construct a single, highly precise standard curve for your target gene. Base it on large number of standards covering a wide concentration range (recommended 7-9 concentrations, each run in 3-4 replicates). This standard curve is then used for interpolation of all field samples. The field samples may be measured over time in independent runs. Proper interpolation then requires that the IPC sample is included in each run. This approach is much more accurate than running a separate standard curve in each run, particularly if based on a smaller number of standards, since the random noise in the separate standard curves introduces systematic run-to-run variation (Tichopad 2012).


Data Analysis - GenEx Software

To enable automatic analysis, runs and interplate calibrators should be indexed in classification columns. It is easy to do this manually. However, if you use pre-plated reactions by leading vendors, the GenEx Software will automatically identify interplate calibrators and annotate your experiment accordingly. A free license for GenEx Enterprise is available for download from www.multid.se and provides the fully functional analysis software for a trial period of 14 days. To purchase GenEx licenses or for qPCR data analysis services, contact us at order@tataa.com.


GenEx is a market-leading software for qPCR experimental design and data processing, and is supported by all leading qPCR instrument manufacturers. It offers user-friendly optimized workflows for qPCR data pre-processing and analysis. Tutorials are available on: www.multid.se/tutorials.php and free support is offered on: www.qpcrforum.com


Manual and product flyer

To download the TATAA IPC manual or product flyer, please visit Product Downloads.


 Contacts and ordering

Place your order here in the webshop or order it by e-mailing order@tataa.com


Shipping costs will apply, please visit General terms and Shipping for more information.

 

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