Technical notes for standard qPCR - Content brought to you by D-Mark Biosciences
In this post, you’ll learn troubleshooting tips and equipment information for qPCR experiments. Quantification and standard curves 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.
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 specificity From 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
*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.
*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.
Mapping out your qPCR experiment
Content brought to you by D-Mar k Biosciences In this post, you’ll learn the basic considerations necessary to design a qPCR experiment.
Measuring RNA with qPCRQuantitative PCR (qPCR), also called real-time PCR, can be used to amplify and simultaneously measure RNA or DNA levels. The process is slightly different depending on whether you’re starting with RNA or DNA. If you’re starting with RNA, you’ll first have to reverse transcribe the RNA into cDNA. You can perform this step separately with kits/reagents designed for reverse transcription, and then proceed to qPCR with your cDNA in the same way you would if you were starting with DNA. Alternatively, there are now kits available, such as PCRBIO’s SyGreen 1-Step Kit, that allow you to proceed through reverse transcription and qPCR in a single step. Measuring DNA or cDNA with qPCRSetting up a qPCR reaction is relatively simple. You’ll provide the DNA or cDNA template and primers that target the specific sequence you want to amplify/measure. The buffer (including salts and dNTP’s), DNA polymerase, and intercalating dye are generally purchased together as part of a kit, such as PCRBIO’s SyGreen Blue Mix.
The productmanual for the kit will specify the recommended concentrations of DNA and primers to use in combination with specified amounts of the kit reagents. When selecting which reagents/kits to use, it is important to consider the detection threshold and the CTvalue. Some kits are designed to be more sensitive to very low concentrations of template, enabling a lower detection threshold. The CTvalue is a measure of how many amplification cycles it takes to reach a detection threshold that is above background. qPCR reagents that enable a lower CTwill enable fewer cycles, and, therefore, less run time. Designing primersand selecting run conditionsYou should keep two factors in mind when designing the primers that will anneal to your template: melting temperature and amplicon length.Melting temperature (TM)is a function of the length and the GC content of your primers. Sequence manipulation softwares such as SnapGene or ApE generally have built in tools for calculating TM, but it can also be determined manually using the equation TM= 2(A+T) + 4(G+C).The reagent kit you are using will generally outline a recommended TM, generally somewhere around 60°C.We recommend the following online tool for primer design: http://bioinfo.ut.ee/primer3-0.4.0/primer3/It’s best to design your experiment, so that your target amplicon is relatively short. As the length of your target amplicon increases, so does the extension time required for each cycle. Therefore, keeping your amplicons short—ideally a few hundred base pairs or less—will decrease the run time of your qPCR experiment.
Additionally, some polymerases and reverse transcriptases are not processive enough to produce very long amplicons. Reagent kit manuals generally also specify recommended time and temperatures for the reverse transcription (if necessary), denaturing, annealing, and extension steps. The primers you designed will help specify the annealing temperature (typically 3-5°Cbelow the TM). The annealing time is a function of the length of the amplicon: recommendations of 1 minute per kilobase are typical. Denaturation typically occurs at 95°C.Keep Reading...qPCR devicesand troubleshootingMeet our favorite qPCR reaction Mix KitsIf you’re starting with RNA: qPCRBIO SyGreen 1-Step KitIf you’re starting with DNA: qPCRBIO SyGreen Blue Mix