Spend your time on research, not on instrument maintenance
Quickly and easily calibrate and verify the performance of your Applied Biosystems real-time PCR system using our instrument-specific kits and plates.
Applied Biosystems spectral calibration kits establish the pure dye spectra and multicomponent values (standards) for each reference dye. These standards enable the real-time PCR system to collect pure spectral information, which it uses algorithmically to help ensure accurate data analysis. Depending on which Applied Biosystems instrument you have, there are plate options for individual dye calibration or multi-dye plates that enable simultaneous calibration, which can greatly reduce the time and effort spent on maintenance.
Confirm the performance of your Applied Biosystems instrument with a TaqMan RNase P instrument verification plate. These plates are pre-loaded with real-time PCR reagents and the TaqMan Assay for the single-copy RNase P gene. Each well contains PCR master mix, RNase P primers, FAM dye-labeled probe, and a known concentration of human genomic DNA template.
Always consult your instrument user guide for the recommended calibration/verification schedule and instructions.
Calibration kits and verification plates by instrument model
Types of calibration
Our calibration kits and plates support several types of calibration.
Region of interest (ROI) calibration
ROI calibration maps the positions of the wells on the sample block of the instrument. The software uses the ROI calibration data to associate increases in fluorescence during a run with the wells on the plate or array card. The instrument uses a set of optical filters to distinguish the fluorescence emissions gathered during runs. The software generates a calibration image for each filter to account for minor differences in the optical path.
Background calibration
Background calibration captures a background image for each optical filter in the absence of sample and reagent and compares the fluorescence from each well to the average for the plate. The software uses that data during subsequent experiments to remove background fluorescence from the experimental data.
Uniformity calibration
Uniformity calibration generates data that allows the software to compensate for the physical effects (non-uniformity of the optical system such as light source) of the instrument filters.
Dye calibration
Dye calibration collects calibration data generated from the dye standards to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the instrument. After each run, the instrument software receives data in the form of a raw spectra signal for each reading. The software determines the contribution of the fluorescent dyes used in each reaction by comparing the raw spectra to the pure spectra calibration data. When you save an experiment after analysis, the instrument software stores the pure spectra with the collected fluorescence data for that experiment.
Calibration helps enable effective multiplexing
To enable success when multiplexing (which helps save on both samples and costs), our custom dye calibration plates help ensure you realize the same level of sensitivity when running multiple targets in a single well as you would running a just one (singleplex). This gene quantification experiment demonstrates comparable results between reactions performed in individual tubes and in 4-plex reactions.
Comparison of singleplex and multiplex gene expression assays
Comparison of singleplex and multiplex gene expression assays. Comparison of singleplex and multiplex gene expression assays. (A) EGFR assay, FAM dye; (B) BRCA1 assay, VIC dye; (C) ESR1 assay, JUN dye; (D) RNase P assay, ABY dye. Amplification was performed on the QuantStudio 7 Real-Time PCR System using TaqMan Multiplex Master Mix. The figure shows amplification plots (left) and linear curves (right) for 4 assays amplified in singleplex (blue) and 4-plex reactions (red) in a dilution series from 20,000 pg to 2 pg of reference colon cDNA per 10 μL reaction. Average Ct value (lines) and average standard deviation (bars) for the dilution series are represented in their respective graphs and show the concordance between singleplex and 4-plex reactions. PCR efficiencies for each target in both single- and multiplex reactions are shown here:
|
EGFR |
BRCA1 |
ESR1 |
RNase P |
|---|---|---|---|---|
Singleplex |
96.09% |
93.56% |
97.13% |
96.91% |
Multiplex (4 targets) |
96.39% |
94.93% |
95.81% |
98.1% |
For Research Use Only. Not for use in diagnostic procedures.