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Pre-designed qRT-PCR primers

qRT-PCR

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#1 moerae

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Posted 31 October 2016 - 05:49 AM

Hi,

 

I was wondering if anyone has any experience with already designed qPCR primers? I've only ever designed my own ones when I was working with zebrafish and the process of testing and validating the primers before I can even use them for the real assay was very laborious and time consuming. I'm currently working with human samples now and OriGene offers predesigned, and I'm going to assume pre-tested, qPCR primers so I'm hoping it will cut down on the time I would need to test and validate the primers.

 

Does anyone have any experience with this kind of thing? If so, how was your experience?

 

Many thanks.



#2 Trof

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Posted 17 November 2016 - 06:56 AM

Unless you use a validated complete assays at set conditions (like ABI/LifeTech Taqmans - but you also need to have all reagents and machine from them) you still need to verfy a predesigned assay, like the OriGene one.

But mostly with human and mouse assays, you just run it once if they work at all (predesigned should), run dilution series, check efficiency, check DNA binding if they are intron spanning, if they are for SYBR you check specificity. That's not that much, finaly you check where the Ct runs in your lowest sample to have an adequate RNA amount and that's all.

But mostly you expect the in silico design to work and to avoid nonspecifities, at least with human and mouse primer designs, there's a large sequence database. It's not that much work for human/mouse assays even if you design them, I would say, can't compere it with other model organisms though.


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