what to do in case of no... (and do you really need them?)

Here we discuss the use and processing of inter-plate calibrators

Moderator: MultiD Support

what to do in case of no... (and do you really need them?)

Postby MultiD Support » Fri Jul 04, 2008 9:58 am

Dear Genexperts,

I have not added IPCs to my plates (I will spare you the details of why).
Now I want to run additional samples on a new plate. My thoughts after reading your manual etc., were that I now do need to calibrate the plates -somehow-... Although I do not understand completely the reasoning behind it: I think that as long as you are able to normalise with your reference genes which are run on the same plate, you end up with a normalised expression of a sample that can just as it is be compared with a normalised expression of a sample that was run on another plate, or am I wrong? (I mean, all of the ct's within a run will be affected the same way by changes in for instance threshold settings or reactionmix)

Than secondly:
What do you think about my idea to calibrate my plates by using the average of all the ct values of my (two) reference genes as an IPC?
Plate setups are in all cases the same (amounts of samples), and I am using the same references in all cases.

Thanks for your advise and help,
also(Mikael) in the genex list! I will get back to that question later and elsewhere in this forum)

Regards,
Muriel de Boer
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Re: what to do in case of no... (and do you really need them?)

Postby MultiD Support » Fri Jul 04, 2008 9:58 am

Muriel,

It is not necessary using interplate calibrators to normalize between plates if:

1) you are using the same reporter for the marker genes and the reference genes (i.e., its OK if both are SYBR assays, but not if you use differently labelled Taqman probes measured in different channels)

and

2) The marker and reference assays have the same PCR efficiencies or you correct for the difference (using GenEx) before normalizing.

Actually, most of these things you can test by generating some data in Excel and testing analyzing them in GenEx with and without interplate calibrators.

If you have two reference genes and they are both good, such that their combined standard deviation is lower than for any one of them you should normalize with both. You can test if this is the case with the Normfinder function in GenEx analyzing all your samples with only the two candidate reference genes.

Good luck!
Mikael
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Re: what to do in case of no... (and do you really need them?)

Postby MultiD Support » Fri Jul 04, 2008 9:59 am

I should add that it is in fact an advantage if one can avoid using interplate calibrators, because every new experimental correction introduced adds to the variation of the measured quantity.

Regards
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Re: what to do in case of no... (and do you really need them

Postby Rosette Bravo » Fri Jan 23, 2015 8:35 am

I am screening 8 samples with many different genes on multiple plates. On each plate I ran two duplicate reactions with one control sample on one control gene as my interplate calibrator (two wells on each plate). How do I enter the calibrator CTs into the data matrix so that I can run interplate calibrator normalization using the data editor tool on Genex?


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Last edited by Rosette Bravo on Wed Feb 04, 2015 9:44 am, edited 1 time in total.
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Re: what to do in case of no... (and do you really need them

Postby MultiDAdmin » Tue Jan 27, 2015 12:27 pm

Hi Rossete

Please have a look at the GenEx example for IPC to see how you should arrange your data. You will find the file in "../GenEx Demo/Data pre-processing" folder.

You can also send me ( amin(at)multid.se ) your data file as MDF and I will help you with that.

BR
Amin Forootan
Multid Support team
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