IPC and reference genes

[Annie]

Dear Dr. Kubista,
I work with RotorGene, currently investigating 30 samples for 3 GOIs with 2 RGs and I cannot use the “All samples” or “All genes” approach. Therefore interplate calibration is very important to me.
I have read your post dated Nov 27, 2009 about interplate calibration, and it was very helpful, however I still have some questions:
1/ Is it possible to run RGs and GOIs on separate plates if interplate calibrator is used, considering it is not possible to fulfill the “All samples” criterion.
2/ my second question is:
If I am not wrong – in one of your post you stated that IPC can be any stable sample (run in triplicate) assayed for any gene (not necessarily GOI or RG in the particular experiment) for each working channel (e.g.FAM). My question is how do I fill the GenEx format if IPC is assayed for a gene different than RG or GOI (or just for GOI for example) Do I need to add another column for this gene? And if so, what should I put for samples in that particular column?
Thank you in advance,
Ana

[Mikael Kubista]

Yes, RG's and GOI's can be run separately if IPC's are used.

See the IPC tutorial on:

http://www.multid.se/tutorials.php

how to arrange data in GenEx.

Good luck!

[Annie]

Dear Dr Kubista,

Thanks a lot for the advice. The tutorials are very helpful.

Regards,
Ana

[Annie]

Dear Dr Kubista,
I really need an advice. I purchased four Taqman Gene Expression assays from Applied Biosystems (two GOI and two RG), and when I validated them on the RotorGene one of the reference genes assays worked well only in 60⁰C (efficiency above 90%), whereas the others only in 57⁰C. My question is:
Would it still be possible to use this reference gene assay using a distinct hybridization temperature if I used an interplate calibrator? Or do I have to purchase new assay to have them all work in 57⁰C?

Lots of thanks in advance and sorry to bother you once again,

Sincerely,
Ana

[Mikael Kubista]

Dear Ana,
In principle you can use assays with different temperature optima in expression analysis assuming of course you run those in separate plates using different temperature protocol. Interplate calibration is not needed since you are automatically using an “all sample” plate out (unless you have so many samples that they do not all fit in the same plate assayed with this marker). However, it is less convenient and may be restrictive.

Good luck!

[Annie]

Thank you very much for your immediate reply.
I do have many samples and I cannot fit them in one run on RotorGene so I guess IPC is a necessity in my case. However, my true concern was the issue of normalizing with reference gene when it is amplified in a different temperature than GOI. I was affraid in such case normalization would not be possible. I wonder what is your opinion on this matter?

Regards,

Ana

[Mikael Kubista]

Ana,
there is no problem in doing that! deltaCq and deltadeltaCq does not assume same temperature protocols.

Good luck!
Mikael

[Annie]

Thanks a lot. That is a relief. I remember you explaining that during the course but it was still a little confusing for me. Now it is clear.

Many thanks and have a great day.

Ana