qPCR stands for Quantitative Real-Time Polymerase Chain Reaction and is a technique which monitors, in real-time, the enzymatic amplification of a targeted sequenceof DNA. Analysis of the resultant amplification curves informs a researcher of the starting amount oftheir DNA.VIEW WORKFLOW PRODUCTS
- Quantification and standard curves
- Efficiency and specificity
- A note on ROX reference dyes
TOOLS & RESOURCES
- Perkin Elmer Chemagic 360
- Macherey-Nagel NucleoMag Pathogen Kits
Technical notes for standard qPCR In this post, you’ll learn troubleshooting tips and equipment information for qPCR experiments.
Relative quantitation can be achieved using an internal reference template. Target quantities can be reported as a fraction of the reference, or the target can be normalized to the reference. For instance, a target that is expected to vary between samples can be compared to a target that is not expected to vary between samples.
Quantification and standard curves
For absolute quantification, a standard curve has to be generated from a series of sample dilutions with known concentrations. For a standard curve, prepare 5-10 serial dilutions that span the highest and lowest levels of target expected. Most qPCR instruments have software for generating a standard curve. If the standard curve is not linear at either the low or high end of sample input, then the assay’s capacity/sensitivity has been exceeded.
Efficiency and specificityFrom the standard curve, the qPCR machine will calculate the efficiency of the reaction. Efficiency is considered to be 100% if the template is amplified 10-fold approximately every 3 cycles.
Efficiencies above 110% suggest that amplification may not be specific.
Melt or dissociation curves can also be used to determine specificity. Melting curves, which are also calculated by typical qPCR instruments, will have a single peak for highly specific interactions. If a reaction has more than one melting curve, then there is more than one template being amplified. Non-specific amplification usually occurs for templates smaller than the target sequence such as primer dimers (primers that have hybridized to each other).
Optimizing and troubleshooting
Optimizing qPCR reaction involves finding the right balance between efficiency and specificity.
Alterations to the reaction conditions that increase specificity may decrease efficiency and vice versa. That means that for a given template and primer combination, you may need to experiment with different primer concentrations and annealing temps.
1) Low Specificity
- reduce primer concentration
- increase annealing temp
- reduce annealing time
- reduce the number of cycles
*If your starting template is RNA, low specificity can also result from carryover of genomic DNA. To make sure that no contaminating DNA persists, it is important to include controls that were not treated with the reverse transcriptase enzyme.
2) Low Efficiency
- increase primer concentration
- decrease annealing temp
- increase annealing time
- increase the number of cycles
*Low efficiency can also result in a late CT (cycle threshold) value. That is, it will take more cycles of amplification for the template to reach a level of fluorescence that is detectable above background. If the fixes listed above do not decrease the CT, primers may need to be redesigned.
See more on primer design for qPCR here.
A note on ROX reference dyes
ROX (carboxy-X-rhodamine) dyes are passive reference dyes sometimes included in qPCR reactions as a reference for fluorescence normalization. The fluorescence intensity is not always measured equally across all wells, particularly for older qPCR machines. The ROX dye, which will be present at the same amount in all wells, provides a baseline fluorescence level that can be used to normalize across wells. Newer machines are designed to minimize the position effects, so different qPCR machines require different levels of ROX.