Optimization of qPCR conditions is important for the development of a robust assay. Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insensitive assays. The two main approaches are optimization of primer concentration and/or annealing temperatures.
Once an assay has been optimized, it is important to verify the reaction efficiency. This information is important when reporting and comparing assays. In this example protocol, the assay efficiency is compared over a wide and narrow dynamic range of cDNA concentrations. In practice, it is common to select a single range to test depending on the expected range of target in the samples, so the protocol given can be adjusted according to the requirements of the experiment. In this example the efficiency is calculated using both 10-fold and 2-fold dilution series. The standard curve should encompass the expected range of expression for the genes of interest Note that the proportionality of the cDNA yield with respect to RNA input is linear when using the ReadyScript® RT kit, so this experiment can be adapted to RT-qPCR if using that system by 1) diluting the RNA and running the RT reactions and 2) then running qPCR on each of the resulting cDNA samples (see Reverse Transcription for examples). However, this is not always the case and does not apply for all Reverse Transcription kits or protocols. This should be verified before adapting this protocol to an alternative kit
1. Prepare a qPCR master mix that is sufficient for 40 reactions following Table P16-40. This allows for extra to
accommodate pipetting errors since 32 reactions will be run (Table P16-41).
2. Dilute the DNA through a series of 1:10 and 1:2 covering 7 dilution points for each series (Table P16-41, Plate Layout for DNA Dilution).
3. Add 5 μL of appropriate template dilution to the defined wells (Table P16-41, Plate Layout for DNA Dilution).
4. Add 15 μL of master mix to each well (Table P16-41).
5. Cap tubes or seal the plate and label. (Make sure the labeling does not obscure instrument excitation/detection
6. Run samples according to the two-step protocol below. Steps 1–2 are repeated through 40 cycles. Follow
amplification with a standard dissociation curve analysis.
Note: Use standard dissociation curve protocol (data collection).