Plate setup???

[eydise]

Hello,
I have 48 well StepOne ABI machine and I have so many samples that I have to run probably 10 plates. I have 3 ref genes and 2 GOI and I want to compare relative expression in control group and treatment group over three time points. I have 2 control groups and 2 treatment groups and 3 biological replicates from each time point from each group. So you see that I have many samples!!! What is the best plate setup for me? And dont I have to include IPC on every plate and also NTC and RT- controls?
Best regards and thanks,
Eydís Elva

[Mikael Kubista]

Dear Eydís,
With a low throughput instrument one must be pragmatic. Best is to use “All-genes” or “All samples” plate designs, because then you don’t need interplate calibration. In your case this is analyzing all samples for GOI_1 on one plate, all samples for GOI_2 on a second plate, RG_1 on a third etc. NTC’s can be run on a separate plate. RG- should be included with the positive samples, but it’s not critical, because the inter-plate calibration is in general only minor, and you only use those samples to have an indication of LOD (when limited by the RT performance).

If you cannot use an “All-samples” design or want to keep flexibility you can include IPC. These should be uncomplicated samples, which robust qPCR assay that give rather low Cq with minimum variation (low SD). You should preferably run the IPC in triplicates. You need one IPC for each channel of the instrument (not one for each assay).

Have you considered outsourcing the expression profiling? TATAA Biocenter (www.tataa.com) , for example, runs customer samples on 384-well plates or even microfluidic cards that take 10000 parallel samples. That would save you time and probably also money (reagent costs are lower on high throughput instruments).

Good luck!

[eydise]

Thank you very much for the reply.....i will consider these options and try to figure something out :)
My best regards,
Eydís

[eydise]

Hi again,
in my assay both of my GOI are coming up with a CT value from 27-30 and I´m using 40 cycles, there are no primers-dimers and no amplification in my NTC og RT- controls. Are these late CT values alright?
Thank you again and my best regards,
Eydís

[Anders Bergkvist]

Hi Eydis,

They may very well be alright. To get a more concrete indicator you may run a gel to check that you get a product of the size you expect. Ultimately to be 100% sure, you may sequence the product.

Best wishes,
Anders

[eydise]

Thank you for the answer :)
Eydís

[Anders Bergkvist]

I assume that by saying that there are no primer-dimers you have checked that with melt curve analysis. This is otherwise a quick and easy way to get an indicator of whether the CT values are valid.

Best wishes,
Anders

[eydise]

Yes I have conducted a melt curve and thats why I say no primer dimer :)
Thanks