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.
Measuring a target quantity relative to one or more stable reference genes using SYBR Green I dye detection is a common application of qPCR. Below is a standard protocol that can be adapted to specific experimental needs.
After optimization of the qPCR assays for both target and reference genes, these are used to measure the quantity of target. A ratio is determined between the quantity of the gene of interest (GOI) and the stable reference gene(s) as described in Data Analysis. In this example, a standard curve is used for determination of copy number. However, a relative quantity can also be determined without a standard curve by using the alternative Comparative Quantification analysis method (Data Analysis). If this approach is adopted, the standard curve is omitted but a calibrator sample is included alongside all test samples. Standard primer concentrations and annealing temperatures are included in the protocol but these should be adapted based on results from optimization experiments (Primer Concentration Optimization and Primer Optimization Using Temperature Gradient).
1. Prepare a different qPCR master mix for each primer pair to be run. Prepare sufficient mix for the samples, standard
curve reactions (described as six dilutions below), No Template Controls, all in duplicate plus calculate an extra 10% to
allow for pipetting error.
For example, if there are five test samples, prepare a mix for samples (5×2) plus standard curve (6×2) plus No Template
Control (NTC) (1×2) = 24 reactions. Therefore prepare a mix for 26.4 or 27 reactions per primer pair.
2. Prepare a 1:10 fold (or suitable dilution for the experiment) serial dilution of suitable standard curve template/cDNA so
that there is 20 μL template of each dilution (six dilutions in total).
3. Add 5 μL of appropriate template serial dilution (standard curve), sample test cDNA or water (NTC) to the defined
tubes or wells.
4. Add 15 μL of master mix to each tube or well.
5. Cap tubes or seal the PCR plate and label. (Make sure the labeling does not obscure the instrument excitation/
detection light path.)
6. Run samples according to Table P17-44.
Note: Use standard dissociation curve protocol (data collection).