New benefits with an “old” method
If you stick to singleplexing but wish to get a variety of information from the same sample
, you have to perform multiple runs. Multiple runs mean more sample material, more reagents, more time. Accuracy, precision and comparability can suffer, as assays are performed in different vessels with different reaction conditions and with a higher risk of pipetting errors.One run with various amplicons instead of several runs
- the advantages of multiplexing are undoubtedly obvious:
Saves precious time for repeated experiment setup/analysis and enhances throughput.
More data from just one sample.
- Saves costs and sample material
Sample material is required only once to amplify all desired target regions. Likewise, PCR-reagents and consumables, which saves your budget.
- Increased accuracy and comparability
Multiplex PCR makes reaction conditions absolutely comparable, because they are identical for each amplicon. Slight differences are not significant as genes to be compared are amplified in the same well.
Pipetting once instead of several times minimizes pipetting errors.
- No investment in a new cycler and consumables
…if you have the right ones!
Putting the cards on the table
Establishing and optimizing the new assay is more complex than a singleplex PCR. Preventing unspecific priming, underrepresented products and loss of yield
are key in assay set-up. Typical measures include: primer design with in silico tools, the avoidance of dNTP-depletion ensured by equally abundant templates, optimizing and matching the individual primer combinations with regard to the PCR conditions. Did you know, that thermocycler and consumables are equally important influencing factors?
The impact of thermocycler and consumables
Cycler and consumables can lead to non-specific PCR products, loss of yield and can affect the reproducibility of results.
Temperature differences and too high or too low temperatures after heating or cooling ramps (over- and undershoot) can reduce PCR yield and increase the risk of mispriming. Plasticizers, biocides or slip agents in plastic-materials can falsify results.
You can avoid that:
1. Guarantee temperature stability & a low over- and undershoot
Precise ramp rates and stable temperatures avoid unspecific amplifications and allow maximum yields. Likewise, the cycler should control that temperatures are within optimal range, i.e. not too high or too low. Temperature stability and precisely controlled temperatures increase the polymerase’s half-life. The enzyme will thank you with a better yield and drastically reduced misprimed secondary products.
2. Go for leachable-free & clean consumables
Ensure to use “clean” PCR-plates, which are free from leachables, nucleases, DNA or PCR-inhibitors. By the way, thin vessel walls guarantee a perfect temperature transfer. To facilitate that more molecules are available for amplification, use plates where fewer molecules adhere (low binding plates).Knowing pitfalls and the way out, establishing a multiplex PCR protocol is not as complex as you might think!
Did we get you hooked?