qPCR nothing worked !!

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varios
varios's picture
qPCR nothing worked !!

 Hi
Im starting a new project with all new samples and genes and starting up a qPCR for validation purposes.

When synthesizing cDNA cDNA I used 600ng (equal amounts of RNA).  
When plating the qPCR i used 2ul of cDNA for a total volume of 20ul per well.
It was a taqman run for 5 genes (two testing genes and 3 housekeeping).

Problem is none of the genes worked in any of the samples.  All CT's were above 40 !!

I will now do it wright and start by doing a standard curve with different cDNA dilutions but i just dont know where too start.
Is my problem caused by very high or very low cDNA concentrations ?? 
Should i start with for example 9ul of cDNA (higher i can go) and down to like 1:2000 or something like that?
Or should I just go from 1:20 to 1:2000 diluted cDNA for example.

Help please !!

Ivan Delgado
Ivan Delgado's picture
When you get zero signal (All

When you get zero signal (All Cts over 40) then there is something going on that simply makes the assays completely fail. My recommendation would be to get a qPCR assay you know works, even if you have to purchase a commercially available one, and make sure your reagents are working by getting a signal with this control assay. If you get this control assay to work then you know the problem is with your assays. In that case you may have to redesign your assays. If this control assay does not work then there is a problem with your cDNA/reagents. In that case you would need to get new reagents and/or get new cDNA synthesized.

varios
varios's picture
 well.  It was all new.  New

 well.  It was all new.  New cDNA reagents, New taqman reagents... and this is my 1st experiment in this lab.... so dont have nay other set that worked before. 

Ivan Delgado
Ivan Delgado's picture
That is why purchasing a qPCR

That is why purchasing a qPCR assay from a commercial source may be the way to go. That way you know with certainty that the assay should work. Of course you could always ask around for someone in your lab, or other labs in your building, that has a qPCR assay they know works well and use that as a control.

varios
varios's picture
Thanks a lot !!...

Thanks a lot !!...
Lets see how it goes !!

eiorns
eiorns's picture
You can outsource your qPCR

You can outsource your qPCR experiment to a core facility at a top US university for free at http://thebenchhq.com. They will design your primers and standard, run your assay with your RNA sample, and analyse your data. It's more cost effective than trouble shooting your experiment yourself (~$12 per sample).