I'm planning to do multiplexing on my qPCRs for 3 different amplicons with widely varying sizes (180 bp, 200 bp and 370 bp) and I need absolute quantification.
Is there going to be a significant bias in the quantification due to the varying sizes? Could you please elaborate your answer and justify it with references or personal data.
Just wondering if anybody has thoughts, theories or experience on such experiment.
(Also asked the same on biology.stackexchange - https://biology.stackexchange.com/questions/79943/multiplex-real-time-pcr-on-different-sized-amplicons)
Safdar Ali could you please elaborate. Do you have experience with multiplex qPCR, are there any cases where it failed, what were the reasons etc.?
Hi,
this is strongly dependent on the used chemistry. From my perspective I would try it with a multiplex qPCR chemistry whihc is designed for difficult multiiplex ing (e.g. you could look which chemistries are capable of 5/6-Plex). The difference in you amplicon sizes is not massive but I would extend annealing extension this will help. So then if you have the chance I would run the 3 Amplicons in one reaction, keep two stable in regard of template and modulate the latter one (e.g. to check linearity etc). This obviously only works in case you have plasmids or so. In general I don't think this is too difficult, as said the used Chemistry is key here
Let me know if this helps and you need more details
Sven
Have used for two amplicons in several cases without any issues. As you would read in the earlier thread about annealing time that's important.
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