The efficiency (E) of a PCR reaction should be 100%, meaning the template doubles after each thermal cycle during exponential amplification. Experimental factors such as the length, secondary structure, and GC content of the amplicon can influence efficiency.
If you choose to design your own real-time PCR primers, take into consideration the following parameters:
- Amplicon length should be approximately 50–150 bp, since longer products do not amplify as efficiently.
- Primers length should be 18–24 nucleotides. This provides for practical annealing temperatures.
- Specific primers– primers should be specific for the target sequence and be free of internal secondary structure. Primers should avoid stretches of homopolymer sequences (e.g., poly(dG)) or repeating motifs, as these can hybridize inappropriately.
- Primer pairs should have compatible melting temperatures (within 1°C).
- Primers’ GC content should be 50%. Primers with high GC content can form stable imperfect hybrids. Conversely, high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3′ end of the primer should be GC rich to enhance annealing of the end that will be extended.
- Primer-dimers -Analyze primer pair sequences to avoid complementarity and hybridization between primers.
- Avoid genomic DNA contamination– For qRT-PCR, design primers that span an exon junction to allow differentiation between amplification of cDNA and potential contaminating genomic DNA by melting curve analysis.
- Confirm the specificity of your primers– Perform a BLAST® search against public databases to be sure that your primers only recognize the target of interest.
- Optimal results may require a titration of primer concentrations between 50 and 500nM. A final concentration of 200 nM for each primer is effective for most reactions.
A reminder for the difference between Sybr based and Taqman based reactions you can find here
Other conditions that may influence efficiency are the dynamics of the reaction itself, the use of non-optimal reagent concentrations, and enzyme quality, which can result in efficiencies below 90%. The presence of PCR inhibitors can produce efficiencies of greater than 110%. A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope of between – 3.58 and – 3.10. The overall efficiency of a real-time PCR reaction depends on the individual efficiencies of the RT reaction and the PCR amplification reaction.
RT efficiency is determined by the percentage of target RNA that is converted into cDNA. PCR amplification efficiency is the most consistent factor in a real-time PCR reaction. However, this amplification exponentially magnifies slight variations in RT efficiency, potentially resulting in misleading data
Reaction efficiency is best assessed through the generation of a standard curve which generated by creating a dilution series of sample nucleic acid and performing real-time PCR. Then, results are plotted with input nucleic acid quantity on the x-axis and Ct on the y-axis. Samples used to generate the standard curve should match (as closely as possible) those that will be used for the experiment (i.e., the same total RNA or DNA sample). The dilution range should span the concentration range expected for the experimental samples. The slope of the curve is used to determine the reaction efficiency.
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Many researchers choose to purchase TaqMan® Assay products and spare the trouble of doing bioinformatics. The assays will all work at Universal Conditions, and they’ll amplify with 100% efficiency with guarantee.
If you want to try out a pre-developed TaqMan® Assay for gene expression, we have excellent coverage for lots of species and for many applications, including SNP genotyping, microRNA and other small RNAs, copy number assays, and more.