Quick Start Guide for qPCR CopyCount

Purpose: This CopyCount Quick Start Guide provides the basic information and best practices for running qPCR CopyCount.

Best practices for setting up your qPCR plate

  1. We recommend that each sample be run with at least 4 replicates. This allows for outliers to be detected and for averaging to improve the quality of your results.
  2. It is best to set up your plate with many different samples and replicates but only a few different assays. We recommend that each 96-well plate contain no more than 4 different assays. Larger plate formats can accommodate more assays.
  3. Perhaps the largest contributor to reducing error is the quality of pipetting. Minimizing random and systematic errors in pipetting is essential to obtaining high quality results. If you are not already familiar with these concepts, please review forward pipetting, reverse pipetting, repetitive pipetting, heterogeneous pipetting, and pipette calibration.

What you need before running qPCR CopyCount

  1. Export the raw data file (in .xls, .xslx, .csv, .txt or .tsv formats ) that contains the fluorescence and cycle-number information. Note that it is very important to submit raw data, not smoothed data (smoothing changes the shape of the qPCR curve and thus corrupts that determination of the copy count).
  2. What is the layout of samples, replicates and assays on your plate? Please see definitions below.
  3. What is your qPCR reaction volume?
  4. Are your target DNAs or RNAs single stranded or double stranded?
  5. What are the names of the assays that are present on your plate?
  6. Has your assay been previously calibrated?

Advantages of qPCR CopyCount

  • Every qPCR well is now an absolute qPCR.
  • No dilution series required
  • No internal or external calibration standards
  • Results are instrument independent and fluorophore independent.
  • Archived qPCR datasets can be analyzed, which enables meta-analysis.
  • cPCR can use TaqMan Probes or Duplex Binding Dyes (however, duplex binding dyes are susceptible to non-specific amplification artifacts).

Limitations of qPCR CopyCount

  • Must use Hot Start PCR to minimize premature amplification and also delayed onset PCR.
  • Will not work with circular plasmid targets (linear plasmids are OK)
  • Will not work with unsheared genomic DNA targets (but does work with sheared genomic DNA).
  • Currently does not work for asymmetric PCR or certain other primer strategies (such as castPCR™, or myT®primers, or competimer™).
  • Getting best results (absolute quantification accuracy of 5% for a single well) requires one-time calibration for each new assay design (i.e. primer set and master mix).
  • Cannot be applied to end-point PCR data

Definitions

Sample: The sample is the biological specimen (human, animal, plant, environment, or other) that contains the target nucleic acid intended for quantification. Typically, the same assay (defined below) will be run on many different samples. The number of assays, samples, and replicates can vary, so please follow the equation below to determine the total number of wells.

Total Wells = Samples x Replicates x Assays

Replicate Set: If two or more wells contain the same sample and the same assay, then those wells form a “replicate set.” Typically, 4 to 96 replicates are run on each sample. The program needs this information to assign which wells should be averaged. Essentially, more replicates means lower error bars.

Why this is important: These replicate sets tell the program which wells should be averaged together to calculate the “Mean Copy Number.” Every well must have assigned to it an assay name and a replicate set name. It is important that the user declare to the program the replicates that correspond to the plate layout that was actually performed. If a well is not declared in any replicate set, then it will be ignored by the program and no copy number will be produced for such undeclared wells. Unused wells should not be declared. Wells with no template controls (NTC) should be declared as a separate replicate set so that the program will appropriately analyze NTCs to determine if any of those wells unexpectedly contain target DNA (i.e. false positives).

Assay: If two qPCR reactions have either a different set of primers or a different master mix, then those reactions are considered to be different assays. A typical qPCR plate will have 1 to 4 different assays. The user needs to provide some information about the assay: is the target double stranded, [primer], [probe], amplicon length, and whether the probe contains an MGB. Each replicate set must have an associated assay name.

Why this is important: qPCR CopyCount uses the assay information to do proper fitting of the curves. If you provide wrong information, it will affect the accuracy of the results.