RMA with biological replicates
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@dipl-ing-johannes-rainer-846
Last seen 10.2 years ago
hi, i have again a question about RMA and how it works with a different number of chips. we are looking at the response of patients to a specific treatment, so we got biological replicates and no technical ones (as we have not enough material from the patients). at the moment i got 9 chips, 3 patients, 3 time points for each patient. What is now the best way to normalize them? normalize them all together with RMA, or normalize the chips from each patient separatly (that means normalize the 3 chips from patient one together, then those from patient 2...)? As i think, RMA gives better results if i have more chips to normalize together (more chips with roughly the same expression result in better fitted model parameters i guess). has anyone a conclusion what's the best was? thanks, jo
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@dipl-ing-johannes-rainer-846
Last seen 10.2 years ago
ok, normalizing all together because of the better model parameter fitting? Quoting Stephen Henderson <s.henderson@ucl.ac.uk>: > all together. > > -----Original Message----- > From: Dipl.-Ing. Johannes Rainer > To: bioconductor@stat.math.ethz.ch > Sent: 7/29/04 2:22 PM > Subject: [BioC] RMA with biological replicates > > hi, > > i have again a question about RMA and how it works with a different > number of > chips. > we are looking at the response of patients to a specific treatment, so > we got > biological replicates and no technical ones (as we have not enough > material > from the patients). at the moment i got 9 chips, 3 patients, 3 time > points for > each patient. > What is now the best way to normalize them? normalize them all together > with > RMA, or normalize the chips from each patient separatly (that means > normalize > the 3 chips from patient one together, then those from patient 2...)? > As i think, RMA gives better results if i have more chips to normalize > together > (more chips with roughly the same expression result in better fitted > model > parameters i guess). > has anyone a conclusion what's the best was? > > thanks, jo > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > ********************************************************************** > This email and any files transmitted with it are confidential and > intended solely for the use of the individual or entity to whom they > are addressed. If you have received this email in error please notify > the system manager (it.support@wibr.ucl.ac.uk). All files are scanned for > viruses. > ********************************************************************** > >
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Perhaps. But my main concern with performing RMA on patient by patient is that the difference between patients could possibly be confounded with normalisation. i.e. You cannot tell if an observed difference is due to patient difference or due to normalisation. The other problem with doing that is that you only have 3 chips. The following thread is almost a year old but it indicates possible problems with only 3 chips. http://files.protsuggest.org/biocond/html/3346.html Regards, Adai. On Thu, 2004-07-29 at 15:13, Dipl.-Ing. Johannes Rainer wrote: > ok, > normalizing all together because of the better model parameter fitting? > > > > Quoting Stephen Henderson <s.henderson@ucl.ac.uk>: > > > all together. > > > > -----Original Message----- > > From: Dipl.-Ing. Johannes Rainer > > To: bioconductor@stat.math.ethz.ch > > Sent: 7/29/04 2:22 PM > > Subject: [BioC] RMA with biological replicates > > > > hi, > > > > i have again a question about RMA and how it works with a different > > number of > > chips. > > we are looking at the response of patients to a specific treatment, so > > we got > > biological replicates and no technical ones (as we have not enough > > material > > from the patients). at the moment i got 9 chips, 3 patients, 3 time > > points for > > each patient. > > What is now the best way to normalize them? normalize them all together > > with > > RMA, or normalize the chips from each patient separatly (that means > > normalize > > the 3 chips from patient one together, then those from patient 2...)? > > As i think, RMA gives better results if i have more chips to normalize > > together > > (more chips with roughly the same expression result in better fitted > > model > > parameters i guess). > > has anyone a conclusion what's the best was? > > > > thanks, jo > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > > > > ********************************************************************** > > This email and any files transmitted with it are confidential and > > intended solely for the use of the individual or entity to whom they > > are addressed. If you have received this email in error please notify > > the system manager (it.support@wibr.ucl.ac.uk). All files are scanned for > > viruses. > > ********************************************************************** > > > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor >
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There may be problems with only 3 arrays. But there should not be a problem with 3 replicates for 3 times = 9 arrays. We used RMA on 2 replicates for 6 treatments = 12 arrays, and the results seemed fine. --Naomi At 03:28 PM 7/29/2004 +0100, Adaikalavan Ramasamy wrote: >Perhaps. But my main concern with performing RMA on patient by patient >is that the difference between patients could possibly be confounded >with normalisation. i.e. You cannot tell if an observed difference is >due to patient difference or due to normalisation. > >The other problem with doing that is that you only have 3 chips. The >following thread is almost a year old but it indicates possible problems >with only 3 chips. > http://files.protsuggest.org/biocond/html/3346.html > >Regards, Adai. > >On Thu, 2004-07-29 at 15:13, Dipl.-Ing. Johannes Rainer wrote: > > ok, > > normalizing all together because of the better model parameter fitting? > > > > > > > > Quoting Stephen Henderson <s.henderson@ucl.ac.uk>: > > > > > all together. > > > > > > -----Original Message----- > > > From: Dipl.-Ing. Johannes Rainer > > > To: bioconductor@stat.math.ethz.ch > > > Sent: 7/29/04 2:22 PM > > > Subject: [BioC] RMA with biological replicates > > > > > > hi, > > > > > > i have again a question about RMA and how it works with a different > > > number of > > > chips. > > > we are looking at the response of patients to a specific treatment, so > > > we got > > > biological replicates and no technical ones (as we have not enough > > > material > > > from the patients). at the moment i got 9 chips, 3 patients, 3 time > > > points for > > > each patient. > > > What is now the best way to normalize them? normalize them all together > > > with > > > RMA, or normalize the chips from each patient separatly (that means > > > normalize > > > the 3 chips from patient one together, then those from patient 2...)? > > > As i think, RMA gives better results if i have more chips to normalize > > > together > > > (more chips with roughly the same expression result in better fitted > > > model > > > parameters i guess). > > > has anyone a conclusion what's the best was? > > > > > > thanks, jo > > > > > > _______________________________________________ > > > Bioconductor mailing list > > > Bioconductor@stat.math.ethz.ch > > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > > > > > > > ********************************************************************** > > > This email and any files transmitted with it are confidential and > > > intended solely for the use of the individual or entity to whom they > > > are addressed. If you have received this email in error please notify > > > the system manager (it.support@wibr.ucl.ac.uk). All files are scanned for > > > viruses. > > > ********************************************************************** > > > > > > > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > >_______________________________________________ >Bioconductor mailing list >Bioconductor@stat.math.ethz.ch >https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor Naomi S. Altman 814-865-3791 (voice) Associate Professor Bioinformatics Consulting Center Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111
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that's ok, but i am afraid that these 3 biological replicates differ to much (three different patients), so that i loose some information when normalizing them all togehter. i think also that the best way to normalize them is to normalizing them all together with RMA. what replicates are you using? technical or biological? you are analyzing 2 replicates exposed to 6 different treatments? Quoting Naomi Altman <naomi@stat.psu.edu>: > There may be problems with only 3 arrays. But there should not be a > problem with 3 replicates for 3 times = 9 arrays. We used RMA on 2 > replicates for 6 treatments = 12 arrays, and the results seemed fine. > > --Naomi > > At 03:28 PM 7/29/2004 +0100, Adaikalavan Ramasamy wrote: > >Perhaps. But my main concern with performing RMA on patient by patient > >is that the difference between patients could possibly be confounded > >with normalisation. i.e. You cannot tell if an observed difference is > >due to patient difference or due to normalisation. > > > >The other problem with doing that is that you only have 3 chips. The > >following thread is almost a year old but it indicates possible problems > >with only 3 chips. > > http://files.protsuggest.org/biocond/html/3346.html > > > >Regards, Adai. > > > >On Thu, 2004-07-29 at 15:13, Dipl.-Ing. Johannes Rainer wrote: > > > ok, > > > normalizing all together because of the better model parameter fitting? > > > > > > > > > > > > Quoting Stephen Henderson <s.henderson@ucl.ac.uk>: > > > > > > > all together. > > > > > > > > -----Original Message----- > > > > From: Dipl.-Ing. Johannes Rainer > > > > To: bioconductor@stat.math.ethz.ch > > > > Sent: 7/29/04 2:22 PM > > > > Subject: [BioC] RMA with biological replicates > > > > > > > > hi, > > > > > > > > i have again a question about RMA and how it works with a different > > > > number of > > > > chips. > > > > we are looking at the response of patients to a specific treatment, so > > > > we got > > > > biological replicates and no technical ones (as we have not enough > > > > material > > > > from the patients). at the moment i got 9 chips, 3 patients, 3 time > > > > points for > > > > each patient. > > > > What is now the best way to normalize them? normalize them all together > > > > with > > > > RMA, or normalize the chips from each patient separatly (that means > > > > normalize > > > > the 3 chips from patient one together, then those from patient 2...)? > > > > As i think, RMA gives better results if i have more chips to normalize > > > > together > > > > (more chips with roughly the same expression result in better fitted > > > > model > > > > parameters i guess). > > > > has anyone a conclusion what's the best was? > > > > > > > > thanks, jo > > > > > > > > _______________________________________________ > > > > Bioconductor mailing list > > > > Bioconductor@stat.math.ethz.ch > > > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > > > > > > > > > > ********************************************************************** > > > > This email and any files transmitted with it are confidential and > > > > intended solely for the use of the individual or entity to whom they > > > > are addressed. If you have received this email in error please notify > > > > the system manager (it.support@wibr.ucl.ac.uk). All files are scanned > for > > > > viruses. > > > > ********************************************************************** > > > > > > > > > > > > > > _______________________________________________ > > > Bioconductor mailing list > > > Bioconductor@stat.math.ethz.ch > > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > > > > > >_______________________________________________ > >Bioconductor mailing list > >Bioconductor@stat.math.ethz.ch > >https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > Naomi S. Altman 814-865-3791 (voice) > Associate Professor > Bioinformatics Consulting Center > Dept. of Statistics 814-863-7114 (fax) > Penn State University 814-865-1348 (Statistics) > University Park, PA 16802-2111 > >
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@james-w-macdonald-5106
Last seen 34 minutes ago
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You will want to run rma on all the chips together. There are not very many instances where it makes sense to run rma on separate batches of chips and then try to combine them. Best, Jim James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 >>> "Dipl.-Ing. Johannes Rainer" <johannes.rainer@tugraz.at> 07/29/04 09:22AM >>> hi, i have again a question about RMA and how it works with a different number of chips. we are looking at the response of patients to a specific treatment, so we got biological replicates and no technical ones (as we have not enough material from the patients). at the moment i got 9 chips, 3 patients, 3 time points for each patient. What is now the best way to normalize them? normalize them all together with RMA, or normalize the chips from each patient separatly (that means normalize the 3 chips from patient one together, then those from patient 2...)? As i think, RMA gives better results if i have more chips to normalize together (more chips with roughly the same expression result in better fitted model parameters i guess). has anyone a conclusion what's the best was? thanks, jo _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
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