Ensure reliable qPCR results—advice on assay validation and data analysis

Your real-time qPCR assay’s accuracy and efficiency is critical for quantification of results

Once qPCR assays have been selected or designed, it is important to validate their accuracy and efficiency to ensure reliable quantification of results. Read about the steps required and use the link to download the Real-time qPCR guide: Part 2—Assay validation and data analysis.

qPCR instrument setup

Once qPCR assays have been selected, or designed (see the Real-time qPCR guide: Part 1—assay design), it is important to validate their accuracy and efficiency to ensure reliable quantification of results. The first step involves making sure your qPCR instrument is setup appropriately to collect and display accurate assay data. This includes:

  • Verifying that instrument channel settings will provide optimal fluorescence calibration and signal normalization.
  • Understanding when to use different data presentation formats (i.e., linear view, normalized linear view, log view).
  • Checking that the baseline is set appropriately. For example, set it in linear view (Rn vs. cycle) to eliminate the background fluorescence seen in early cycles of amplification. However, baseline should not overlap with the area in which the amplification signal begins to rise above background.
  • Ensuring that the threshold is set above the baseline and through the amplification curve in the exponential growth region (during the linear increasing signal in a log scale).

Section 2, Instrument reporting, of the Real-time qPCR Guide: Part 2—assay validation and data analysis discusses these recommendations and provides a general calibration protocol.

Assay validation

Before you use valuable experimental samples in a qPCR experiment, evaluate your assays for their specificity and efficiency. It is especially important to validate any new assays to confirm they are performing according to the expected standards under the experimental conditions you plan to use.

  • Ensure signal is coming from the expected target; rule out nonspecific amplification. Consider melt curve results, amplicon size, and sequence of the PCR product.
  • Calculate reaction efficiency using a standard curve.
  • Perform a standard curve for each assay.Retest newly made standards and controls.

Section 3, Assay validation, discusses methods for determining assay specificity, calculating reaction efficiency and the factors that affect it, and the setup of standard curves using reference genes.

Data analysis

After running your qPCR experiment, use the amplification curve data to determine the expression level of your target sequence. Section 4, Data analysis, provides general guidelines for qPCR data analysis with examples of both absolute and relative quantification to measure changes in gene expression.

Additional qPCR resources

Section 5, Additional qPCR resources, describes several free, online tools and calculators that can aid in qPCR assay design and setup. Links to their access are provided. There are also links to several informative webinars and educational articles, including tips for multiplex experiments, data normalization, and MIQE recommendations for qPCR data reporting. IDT has over 60 educational articles and 30 webinars on qPCR topics alone.

Further, detailed guidance on assay validation and data analysis

All of the above points are examined in detail with best practice recommendations and expertise from our research and development scientists in the Real-time qPCR Guide: Part 2—assay validation and data analysis, a follow-up to the Real-time qPCR guide: Part 1—assay design. Obtain it free by simply registering for it here.

Published Nov 5, 2019