What to do first?

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

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What to do first?

Postby Annie » Thu Mar 10, 2011 10:01 pm

Dear Mr Kubista,

I have two questions about data pre-processing and I would really appreciate your help.

1. I have different amounts of RT product for different samples (left from previous experiment) that I would like to use in my study. I will need to dilute some samples more than others In order to run all the genes I plan. Can I use the "normalization to sample amount" option in GenEx during data pre-processing to normalize for such differences. I will have different (although not exactly known) cDNA amounts. Basing on RNA input in RT reactions and assuming 100% RT efficiency I can count the input of cDNA in every PCR reaction. Can I put it in the column named #total RNA?

2. I did a study of 31 genes in 50 patients. I used two different assays as IPCs (one for green and one for yellow channel and different than my studied genes or RGs) which were run in a commercially available cDNA. cDNA input in IPCs was different than that of the samples. My question is: what should I do first during data pre-processing inter-plate calibration or normalization to sample amount?

I will greatly appreciate your help,
Thank you in advance,

Ania
Annie
 
Posts: 18
Joined: Fri Jun 25, 2010 10:06 am

Re: What to do first?

Postby Mikael Kubista » Thu Mar 10, 2011 10:30 pm

Dear Ania,
Yes, you can use the GenEx option to normalize to sample amount. Note, though, if you normalize with reference genes later, this neutralizes the normalization to sample amount.

The order of IPC and normalization with sample amount does not matter - try it; its so easy to test with GenEx!

Note, if you perform pre-processing following the steps from top to bottom in the GenEx Editor pre-processing menu you get right.

Good luck
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm

Re: What to do first?

Postby Annie » Thu Mar 10, 2011 11:13 pm

Thank you so much for such a quick response.

I know that normalization with reference genes would normalize most differences, but in my case I have three RT replicates for each sample and two PCR replicates. In case of some samples I have different volumes of each of the three RTs. Could this influence final results if I did not normalize to sample amount? And, should I normalize to RGs first and then avarage RT replicates?

My question about sequence of normalization steps in pre-processing was due to the fact that when couple of months ago I e-mailed my data to Mrs. Johanna from TATAA to check if they were correctly put for GenEx format I got a response from her which suggested to normalize first to RNA, before IPC normalization. It was stated in the e-mail that if normalized first with IPCs, all wells would be corrected in the same way although they contained different amounts of RNA. That is why I thought it might matter, plus I was worried about different sample input in IPCs.

Once again lots of thanks and have a nice evening,

Ania
Annie
 
Posts: 18
Joined: Fri Jun 25, 2010 10:06 am

Re: What to do first?

Postby Mikael Kubista » Fri Mar 11, 2011 2:49 pm

Assuming the RT reaction reverse transcribes all genes, than normalization with reference gene(-s) corrects for any variation in RT volumes. Only if you run 1-step RT-qPCR volume normalization is needed.

Good luck
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm

Re: What to do first?

Postby Annie » Sun Mar 13, 2011 8:54 pm

Thank you very much for your answer.

Regards,

Ania
Annie
 
Posts: 18
Joined: Fri Jun 25, 2010 10:06 am


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