I have conflicting recommendations from Fluidigm tech support and users as to whether it is worth it to run each gene with two sets of primers. I am running a 96.96 gene expression array on multiple mouse tissues. With wet lab testing available, it seems like overkill to use two sets for each gene. But because each chip is so expensive, it might be worth it to investigate my top genes of interest more thoroughly. Any recommendations?
Edit: My colleague showed me several examples on his own chip where primers from Fluidigm had lower Ct values than expected whereas primers he designed himself for the same gene showed the expected expression. While some of the replicate primer sets matched exactly, the enhanced signal from Fluidigm primers was observed for multiple genes. The efficiency of all primers was acceptable.
Considering that the chips are quite expensive, I do think that it would be beneficial to run each gene with 2 sets of primers. Good luck!
Presuming your primers have already been tested on another qPCR instrument and you're satisfied with their efficiency (and they produce only your intended signal), I can't see why you need the redundancy. I would rather have data for another gene rather than duplicate data taking up space on the chip.
I updated my discussion above to include details about why I am considering two primer sets for each gene. Has anyone else observed enhanced expression with the chip or with Fluidigm primers?
If you have already checked your primer with another qPCR instrument, you can go for another gene instead of 2nd set of primer as the chip is expensive. I have also seen that Fluidigm has lower Ct values than expected.
Thanks for the suggestions. I ended up choosing two primer sets for each gene, because every gene was of high interest, and none had been validated with a different qPCR method. For a broader screen, I would go with one set per gene.
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