Question: Pooling in microarray studies
0
gravatar for Wiesner Vos
16.1 years ago by
Wiesner Vos50
Wiesner Vos50 wrote:
I have question arising to the pooling of mRNA samples. Someone approached me about the following problem: The study wants to use Affymetrix chips to study changes in expression between a group of treated mice and a group untreated mice. There are 10 mice in each group. It is only possible to extract 8 ug of RNA from each mouse, not enough for one chip. (According to the experimenters they require 10 ug per chip) So it is not possible to use biological replicate chips for each individual mice. Now the issue is whether to perhaps pool the RNA in each group and carry out analysis on technical replicates from the pooled samples. As I understand it pooling may reduce the precision, with the risk that one or few samples can dominate the outcome, and that averaging over single sample hybridisations is perhaps safer than using pooled samples. However in this case you cannot do single sample hybridisations. I was wondering if the following approach is an acceptable compromise to retain at least some information on the between sample variation in each group: Mix the RNA from 2 different mice on a single chip to get 5 hybridisations, where the hybridisation on each chip is from the mix of the RNA samples of two mice? I though that this may enable you to some extend if all the mice are behaving similarly. Ofcourse one would not be able to distinguish between the behaviour of the two mice relating to the same chip. Or is it better to accept that you do not have enough RNA to hybridize the sample for each individual to a separate chip and pool the samples and accept the risk that one sample may dominate the outcome? The best solution did not seem obvious (to me at least!) Any comments will be much appreciated. Wiesner Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom
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ADD COMMENTlink modified 16.1 years ago by Tan, MinHan180 • written 16.1 years ago by Wiesner Vos50
Answer: Pooling in microarray studies
0
gravatar for James W. MacDonald
16.1 years ago by
United States
James W. MacDonald51k wrote:
You might consider trying to run the chips with 8 ug mRNA anyway. At our facility we routinely run chips with as little as 5 ug. 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 >>> Wiesner Vos <vos@stats.ox.ac.uk> 10/06/03 09:27AM >>> I have question arising to the pooling of mRNA samples. Someone approached me about the following problem: The study wants to use Affymetrix chips to study changes in expression between a group of treated mice and a group untreated mice. There are 10 mice in each group. It is only possible to extract 8 ug of RNA from each mouse, not enough for one chip. (According to the experimenters they require 10 ug per chip) So it is not possible to use biological replicate chips for each individual mice. Now the issue is whether to perhaps pool the RNA in each group and carry out analysis on technical replicates from the pooled samples. As I understand it pooling may reduce the precision, with the risk that one or few samples can dominate the outcome, and that averaging over single sample hybridisations is perhaps safer than using pooled samples. However in this case you cannot do single sample hybridisations. I was wondering if the following approach is an acceptable compromise to retain at least some information on the between sample variation in each group: Mix the RNA from 2 different mice on a single chip to get 5 hybridisations, where the hybridisation on each chip is from the mix of the RNA samples of two mice? I though that this may enable you to some extend if all the mice are behaving similarly. Ofcourse one would not be able to distinguish between the behaviour of the two mice relating to the same chip. Or is it better to accept that you do not have enough RNA to hybridize the sample for each individual to a separate chip and pool the samples and accept the risk that one sample may dominate the outcome? The best solution did not seem obvious (to me at least!) Any comments will be much appreciated. Wiesner Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
ADD COMMENTlink written 16.1 years ago by James W. MacDonald51k
Answer: Pooling in microarray studies
0
gravatar for Tan, MinHan
16.1 years ago by
Tan, MinHan180
Tan, MinHan180 wrote:
- Perhaps it is an policy issue with your microarray core facility, but the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug total RNA. - I routinely use 5 ug total RNA with no apparent problems. - So yes, you should be able to do single sample hybridizations. Regards, Min-Han -----Original Message----- From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] Sent: Monday, October 06, 2003 9:27 AM To: bioconductor@stat.math.ethz.ch Subject: [BioC] Pooling in microarray studies I have question arising to the pooling of mRNA samples. Someone approached me about the following problem: The study wants to use Affymetrix chips to study changes in expression between a group of treated mice and a group untreated mice. There are 10 mice in each group. It is only possible to extract 8 ug of RNA from each mouse, not enough for one chip. (According to the experimenters they require 10 ug per chip) So it is not possible to use biological replicate chips for each individual mice. Now the issue is whether to perhaps pool the RNA in each group and carry out analysis on technical replicates from the pooled samples. As I understand it pooling may reduce the precision, with the risk that one or few samples can dominate the outcome, and that averaging over single sample hybridisations is perhaps safer than using pooled samples. However in this case you cannot do single sample hybridisations. I was wondering if the following approach is an acceptable compromise to retain at least some information on the between sample variation in each group: Mix the RNA from 2 different mice on a single chip to get 5 hybridisations, where the hybridisation on each chip is from the mix of the RNA samples of two mice? I though that this may enable you to some extend if all the mice are behaving similarly. Ofcourse one would not be able to distinguish between the behaviour of the two mice relating to the same chip. Or is it better to accept that you do not have enough RNA to hybridize the sample for each individual to a separate chip and pool the samples and accept the risk that one sample may dominate the outcome? The best solution did not seem obvious (to me at least!) Any comments will be much appreciated. Wiesner Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you.
ADD COMMENTlink written 16.1 years ago by Tan, MinHan180
Thanks for the replies Min-han and Jim. I suspected that this may be the case (keeping the papers in mind about comparing expression measures on the Dilution data eg. Cope et al.) I suspected that the use 8ug would be perhaps not make much of a difference if you use an expression measure like for example RMA that seems to be reasonably independent of the actual amount of RNA on a chip. Thanks again for your help. Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom On Mon, 6 Oct 2003, Tan, MinHan wrote: > - Perhaps it is an policy issue with your microarray core facility, but > the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug > total RNA. > > - I routinely use 5 ug total RNA with no apparent problems. > > - So yes, you should be able to do single sample hybridizations. > > Regards, > Min-Han > > > > -----Original Message----- > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > Sent: Monday, October 06, 2003 9:27 AM > To: bioconductor@stat.math.ethz.ch > Subject: [BioC] Pooling in microarray studies > > > > I have question arising to the pooling of mRNA > samples. Someone approached me about the > following problem: > > The study wants to use Affymetrix chips to study > changes in expression between a group of treated > mice and a group untreated mice. There are 10 mice > in each group. It is only possible to extract > 8 ug of RNA from each mouse, not enough for one chip. (According to the > experimenters they require 10 ug per > chip) So it is not possible to use biological > replicate chips for each individual mice. Now the issue > is whether to perhaps pool the RNA in each group > and carry out analysis on technical replicates from the > pooled samples. > > As I understand it pooling may reduce the precision, with > the risk that one or few samples can dominate the outcome, and that > averaging over single sample hybridisations is perhaps safer than using > pooled samples. However in this case you cannot do single sample > hybridisations. > > I was wondering if the following approach is an acceptable compromise to > retain at least some information on the between sample variation in each > group: > > Mix the RNA from 2 different mice on a single chip to get 5 > hybridisations, where the hybridisation on each chip is from the mix of > the RNA samples of two mice? I though that this may enable you to some > extend if all the mice are behaving similarly. Ofcourse one would not be > able to distinguish between the behaviour of the two mice relating to > the same chip. Or is it better to accept that you do not have enough RNA > to hybridize the sample for each individual to a separate chip and pool > the samples and accept the risk that one sample may dominate the > outcome? The best solution did not seem obvious (to me at least!) > > Any comments will be much appreciated. > > Wiesner > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor >
ADD REPLYlink written 16.1 years ago by Wiesner Vos50
>From a study I have done on affymatrix chips (HU95A). the pooled sample does reproduce the average of individual samples. I haven't got time to write the paper. but I will be happen to sent you the data that demonstrate this. By pooling, the biological variation are averaged, the only power you lose is from the technical variation, which is quite low with affymatrix chips. As I know, similar but larger studies on pooling is undergone at U. Of Wisconsin on mice and rats. I expect the similar results will be shown. Pooling, however, is more suitable for mice studies(animal in general) than human, as the mices being pooled are often similar from same strain. Also, pooling should have some other advantage in your case as the signal/noise ratio should be higher for pooled sample (more RNA) compared to the individuals. Kenny Kenny Ye Assistant Professor Department of Applied Math and Statistics SUNY at Stony Brook Stony Brook, New York 11794-3600 Phone (631)632-9344 Fax (631)632-8490 On Mon, 6 Oct 2003, Wiesner Vos wrote: > > Thanks for the replies Min-han and Jim. > I suspected that this may be the case (keeping the > papers in mind about comparing expression measures > on the Dilution data eg. Cope et al.) > > I suspected that the use 8ug would be perhaps > not make much of a difference if you use > an expression measure like for example > RMA that seems to be reasonably independent of > the actual amount of RNA on a chip. Thanks > again for your help. > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > > > > > > On Mon, 6 Oct 2003, Tan, MinHan wrote: > > > - Perhaps it is an policy issue with your microarray core facility, but > > the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug > > total RNA. > > > > - I routinely use 5 ug total RNA with no apparent problems. > > > > - So yes, you should be able to do single sample hybridizations. > > > > Regards, > > Min-Han > > > > > > > > -----Original Message----- > > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > > Sent: Monday, October 06, 2003 9:27 AM > > To: bioconductor@stat.math.ethz.ch > > Subject: [BioC] Pooling in microarray studies > > > > > > > > I have question arising to the pooling of mRNA > > samples. Someone approached me about the > > following problem: > > > > The study wants to use Affymetrix chips to study > > changes in expression between a group of treated > > mice and a group untreated mice. There are 10 mice > > in each group. It is only possible to extract > > 8 ug of RNA from each mouse, not enough for one chip. (According to the > > experimenters they require 10 ug per > > chip) So it is not possible to use biological > > replicate chips for each individual mice. Now the issue > > is whether to perhaps pool the RNA in each group > > and carry out analysis on technical replicates from the > > pooled samples. > > > > As I understand it pooling may reduce the precision, with > > the risk that one or few samples can dominate the outcome, and that > > averaging over single sample hybridisations is perhaps safer than using > > pooled samples. However in this case you cannot do single sample > > hybridisations. > > > > I was wondering if the following approach is an acceptable compromise to > > retain at least some information on the between sample variation in each > > group: > > > > Mix the RNA from 2 different mice on a single chip to get 5 > > hybridisations, where the hybridisation on each chip is from the mix of > > the RNA samples of two mice? I though that this may enable you to some > > extend if all the mice are behaving similarly. Ofcourse one would not be > > able to distinguish between the behaviour of the two mice relating to > > the same chip. Or is it better to accept that you do not have enough RNA > > to hybridize the sample for each individual to a separate chip and pool > > the samples and accept the risk that one sample may dominate the > > outcome? The best solution did not seem obvious (to me at least!) > > > > Any comments will be much appreciated. > > > > Wiesner > > > > > > > > Wiesner J. Vos > > Department of Statistics > > University of Oxford > > 1 South Parks Road > > OX1 3TG > > United Kingdom > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. > > > > _______________________________________________ > > 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 >
ADD REPLYlink written 16.1 years ago by Kenny Ye100
Answer: Pooling in microarray studies
0
gravatar for James W. MacDonald
16.1 years ago by
United States
James W. MacDonald51k wrote:
What you need to have is 16 ug of fragmented cRNA to hybridize to the chip. If you start with 10 ug, there is a very high probability that you will end up with at least 16 ug at the end of the molecular biology step. However, if you only start with 8 ug, you are less likely to end up with the magical 16 ug, but it is still highly probable. We recently did something like 8 chips with only ~5 ug for each, and had 16 ug at the end for all of them. 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 >>> Wiesner Vos <vos@stats.ox.ac.uk> 10/06/03 10:02AM >>> Thanks for the replies Min-han and Jim. I suspected that this may be the case (keeping the papers in mind about comparing expression measures on the Dilution data eg. Cope et al.) I suspected that the use 8ug would be perhaps not make much of a difference if you use an expression measure like for example RMA that seems to be reasonably independent of the actual amount of RNA on a chip. Thanks again for your help. Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom On Mon, 6 Oct 2003, Tan, MinHan wrote: > - Perhaps it is an policy issue with your microarray core facility, but > the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug > total RNA. > > - I routinely use 5 ug total RNA with no apparent problems. > > - So yes, you should be able to do single sample hybridizations. > > Regards, > Min-Han > > > > -----Original Message----- > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > Sent: Monday, October 06, 2003 9:27 AM > To: bioconductor@stat.math.ethz.ch > Subject: [BioC] Pooling in microarray studies > > > > I have question arising to the pooling of mRNA > samples. Someone approached me about the > following problem: > > The study wants to use Affymetrix chips to study > changes in expression between a group of treated > mice and a group untreated mice. There are 10 mice > in each group. It is only possible to extract > 8 ug of RNA from each mouse, not enough for one chip. (According to the > experimenters they require 10 ug per > chip) So it is not possible to use biological > replicate chips for each individual mice. Now the issue > is whether to perhaps pool the RNA in each group > and carry out analysis on technical replicates from the > pooled samples. > > As I understand it pooling may reduce the precision, with > the risk that one or few samples can dominate the outcome, and that > averaging over single sample hybridisations is perhaps safer than using > pooled samples. However in this case you cannot do single sample > hybridisations. > > I was wondering if the following approach is an acceptable compromise to > retain at least some information on the between sample variation in each > group: > > Mix the RNA from 2 different mice on a single chip to get 5 > hybridisations, where the hybridisation on each chip is from the mix of > the RNA samples of two mice? I though that this may enable you to some > extend if all the mice are behaving similarly. Ofcourse one would not be > able to distinguish between the behaviour of the two mice relating to > the same chip. Or is it better to accept that you do not have enough RNA > to hybridize the sample for each individual to a separate chip and pool > the samples and accept the risk that one sample may dominate the > outcome? The best solution did not seem obvious (to me at least!) > > Any comments will be much appreciated. > > Wiesner > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. > > _______________________________________________ > 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
ADD COMMENTlink written 16.1 years ago by James W. MacDonald51k
Answer: Pooling in microarray studies
0
gravatar for Crispin Miller
16.1 years ago by
Crispin Miller1.1k
Crispin Miller1.1k wrote:
Our experience is that it's really important to be consistent - if you use 8ug for one sample use 8ug for all the rest... crispin > -----Original Message----- > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > Sent: 06 October 2003 15:02 > To: Tan, MinHan > Cc: bioconductor@stat.math.ethz.ch > Subject: RE: [BioC] Pooling in microarray studies > > > > Thanks for the replies Min-han and Jim. > I suspected that this may be the case (keeping the > papers in mind about comparing expression measures > on the Dilution data eg. Cope et al.) > > I suspected that the use 8ug would be perhaps > not make much of a difference if you use > an expression measure like for example > RMA that seems to be reasonably independent of > the actual amount of RNA on a chip. Thanks > again for your help. > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > > > > > > On Mon, 6 Oct 2003, Tan, MinHan wrote: > > > - Perhaps it is an policy issue with your microarray core > facility, but > > the standard Affymetrix GeneChip Technical Manual > recommends 5 - 20 ug > > total RNA. > > > > - I routinely use 5 ug total RNA with no apparent problems. > > > > - So yes, you should be able to do single sample hybridizations. > > > > Regards, > > Min-Han > > > > > > > > -----Original Message----- > > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > > Sent: Monday, October 06, 2003 9:27 AM > > To: bioconductor@stat.math.ethz.ch > > Subject: [BioC] Pooling in microarray studies > > > > > > > > I have question arising to the pooling of mRNA > > samples. Someone approached me about the > > following problem: > > > > The study wants to use Affymetrix chips to study > > changes in expression between a group of treated > > mice and a group untreated mice. There are 10 mice > > in each group. It is only possible to extract > > 8 ug of RNA from each mouse, not enough for one chip. > (According to the > > experimenters they require 10 ug per > > chip) So it is not possible to use biological > > replicate chips for each individual mice. Now the issue > > is whether to perhaps pool the RNA in each group > > and carry out analysis on technical replicates from the > > pooled samples. > > > > As I understand it pooling may reduce the precision, with > > the risk that one or few samples can dominate the outcome, and that > > averaging over single sample hybridisations is perhaps > safer than using > > pooled samples. However in this case you cannot do single sample > > hybridisations. > > > > I was wondering if the following approach is an acceptable > compromise to > > retain at least some information on the between sample > variation in each > > group: > > > > Mix the RNA from 2 different mice on a single chip to get 5 > > hybridisations, where the hybridisation on each chip is > from the mix of > > the RNA samples of two mice? I though that this may enable > you to some > > extend if all the mice are behaving similarly. Ofcourse one > would not be > > able to distinguish between the behaviour of the two mice > relating to > > the same chip. Or is it better to accept that you do not > have enough RNA > > to hybridize the sample for each individual to a separate > chip and pool > > the samples and accept the risk that one sample may dominate the > > outcome? The best solution did not seem obvious (to me at least!) > > > > Any comments will be much appreciated. > > > > Wiesner > > > > > > > > Wiesner J. Vos > > Department of Statistics > > University of Oxford > > 1 South Parks Road > > OX1 3TG > > United Kingdom > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > > This email message, including any attachments, is for the > sole use of the intended recipient(s) and may contain > confidential information. Any unauthorized review, use, > disclosure or distribution is prohibited. If you are not the > intended recipient(s) please contact the sender by reply > email and destroy all copies of the original message. Thank you. > > > > _______________________________________________ > > 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 > -------------------------------------------------------- This email is confidential and intended solely for the use o...{{dropped}}
ADD COMMENTlink written 16.1 years ago by Crispin Miller1.1k
Answer: Pooling in microarray studies
0
gravatar for Dapeng Cui
16.1 years ago by
Dapeng Cui40
Dapeng Cui40 wrote:
To my experience on mouse tissue, 5ug total RNA --> 5ug cDNA --> 50~150ug cRNA, enough for five chips. If I have less than 0.5ug total RNA I'll run a double labelling, which gives me 20~40ug cRNA. Of course you need to keep the the starting amount consistent for all samples. dapeng >>> "James MacDonald" <jmacdon@med.umich.edu> 10/06/03 10:33AM >>> What you need to have is 16 ug of fragmented cRNA to hybridize to the chip. If you start with 10 ug, there is a very high probability that you will end up with at least 16 ug at the end of the molecular biology step. However, if you only start with 8 ug, you are less likely to end up with the magical 16 ug, but it is still highly probable. We recently did something like 8 chips with only ~5 ug for each, and had 16 ug at the end for all of them. 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 >>> Wiesner Vos <vos@stats.ox.ac.uk> 10/06/03 10:02AM >>> Thanks for the replies Min-han and Jim. I suspected that this may be the case (keeping the papers in mind about comparing expression measures on the Dilution data eg. Cope et al.) I suspected that the use 8ug would be perhaps not make much of a difference if you use an expression measure like for example RMA that seems to be reasonably independent of the actual amount of RNA on a chip. Thanks again for your help. Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom On Mon, 6 Oct 2003, Tan, MinHan wrote: > - Perhaps it is an policy issue with your microarray core facility, but > the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug > total RNA. > > - I routinely use 5 ug total RNA with no apparent problems. > > - So yes, you should be able to do single sample hybridizations. > > Regards, > Min-Han > > > > -----Original Message----- > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > Sent: Monday, October 06, 2003 9:27 AM > To: bioconductor@stat.math.ethz.ch > Subject: [BioC] Pooling in microarray studies > > > > I have question arising to the pooling of mRNA > samples. Someone approached me about the > following problem: > > The study wants to use Affymetrix chips to study > changes in expression between a group of treated > mice and a group untreated mice. There are 10 mice > in each group. It is only possible to extract > 8 ug of RNA from each mouse, not enough for one chip. (According to the > experimenters they require 10 ug per > chip) So it is not possible to use biological > replicate chips for each individual mice. Now the issue > is whether to perhaps pool the RNA in each group > and carry out analysis on technical replicates from the > pooled samples. > > As I understand it pooling may reduce the precision, with > the risk that one or few samples can dominate the outcome, and that > averaging over single sample hybridisations is perhaps safer than using > pooled samples. However in this case you cannot do single sample > hybridisations. > > I was wondering if the following approach is an acceptable compromise to > retain at least some information on the between sample variation in each > group: > > Mix the RNA from 2 different mice on a single chip to get 5 > hybridisations, where the hybridisation on each chip is from the mix of > the RNA samples of two mice? I though that this may enable you to some > extend if all the mice are behaving similarly. Ofcourse one would not be > able to distinguish between the behaviour of the two mice relating to > the same chip. Or is it better to accept that you do not have enough RNA > to hybridize the sample for each individual to a separate chip and pool > the samples and accept the risk that one sample may dominate the > outcome? The best solution did not seem obvious (to me at least!) > > Any comments will be much appreciated. > > Wiesner > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. > > _______________________________________________ > 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 _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
ADD COMMENTlink written 16.1 years ago by Dapeng Cui40
Answer: Pooling in microarray studies
0
gravatar for Christina Kendziorski
16.1 years ago by
Christina Kendziorski60 wrote:
Hi Wiesner, Pooling, as you have noted, is often done when sufficient mRNA is not available. I see that others have indicated that you probably have enough mRNA...but I will make a few comments anyway. Pooling is also done in an effort to reduce the effect of biological variability. There are many studies that have used pooled samples for this latter purpose. Provided certain assumptions hold (mRNAs average out when pooled and there are no outliers), pooling can be advantageous. You end up getting an estimate of the sum of pool to pool and technical variability.... and provided you are making inferences about pools of subjects (as opposed to individuals - which is often the case when studying experimental populations), this estimate is the one you need. I discuss this in a recent Biostatistics paper. I'll send it under separate cover. We are doing studies to check these assumptions using ~60 Affy chips. There is some evidence to think that the assumptions might NOT hold. Finally, I will note that if there is contamination (let's say an outlier animal), pooling might still be useful. As I argued at a talk at JSM (slides are at my website), there are different cases of contamination to consider. THe most realistic one is that an animal is an outlier in say some collection of genes....and you don't know of course what that collection is. You'll either end up averaging at the mRNA level (again, provided mRNAs average) or you will end up averaging the individual measurements across arrays (after normalizing). I hope that helps. Christina Christina Kendziorski Assistant Professor Department of Biostatistics and Medical Informatics University of Wisconsin - Madison Medical Sciences Center (6729) 1300 University Avenue Madison, Wisconsin 53706 Phone: (608) 262-3146 Fax: (608) 265-7916 > I have question arising to the pooling of mRNA > samples. Someone approached me about the > following problem: > > The study wants to use Affymetrix chips to study > changes in expression between a group of treated > mice and a group untreated mice. There are 10 mice > in each group. It is only possible to extract > 8 ug of RNA from each mouse, not enough for one chip. > (According to the experimenters they require 10 ug per > chip) So it is not possible to use biological > replicate chips for each individual mice. Now the issue > is whether to perhaps pool the RNA in each group > and carry out analysis on technical replicates from the > pooled samples. > > As I understand it pooling may reduce the precision, with > the risk that one or few samples can dominate the outcome, and > that averaging over single sample hybridisations is perhaps > safer than using pooled samples. However in this case you cannot > do single sample hybridisations. > > I was wondering if the following approach is an acceptable > compromise to retain at least some information on the between > sample variation in each group: > > Mix the RNA from 2 different mice on a single chip to get 5 > hybridisations, where the hybridisation on each chip is from the > mix of the RNA samples of two mice? I though that this may > enable you to some extend if all the mice are behaving > similarly. Ofcourse one would not be able to distinguish > between the behaviour of the two mice relating to the same > chip. Or is it better to accept that you do not have enough > RNA to hybridize the sample for each individual to a separate > chip and pool the samples and accept the risk that > one sample may dominate the outcome? The best > solution did not seem obvious (to me at least!) > > Any comments will be much appreciated. > > Wiesner > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom
ADD COMMENTlink written 16.1 years ago by Christina Kendziorski60
Answer: Pooling in microarray studies
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gravatar for Tan, MinHan
16.1 years ago by
Tan, MinHan180
Tan, MinHan180 wrote:
- I personally have not had the experience of <30 ug cRNA with 5 ug total RNA. (I adhere rather painstakingly - almost mule-headedly - to the protocol, but anyone might too, if one was operating with $5000 worth of chips/reageants at one go. ;P) - Also, the amount used for the IVT reaction, even for starting amount of 5 ug total RNA, is only about 10 uL of the 12 uL from cDNA synthesis reaction/cleanup, so one can assume that there would be even more cRNA synthesized if I used the entire cDNA synthesis reaction. - I am curious - why is there a need to assume equal amounts of starting total RNA, if the loading amount of cRNA is the same? Wouldn't normalization manage any minor differences in loading cRNA? The amount loaded on the HGU133A chip is fixed at about 10 ug cRNA (although the Affymetrix protocol makes you jump through hoops to calculate that) (since the hybridization amount is 15 ug, but only 200 uL out of 300 uL of hybridization mixture is used for the GeneChip) Regards, Min-Han -----Original Message----- From: James MacDonald [mailto:jmacdon@med.umich.edu] Sent: Monday, October 06, 2003 10:33 AM To: vos@stats.ox.ac.uk; Tan, MinHan Cc: bioconductor@stat.math.ethz.ch Subject: RE: [BioC] Pooling in microarray studies What you need to have is 16 ug of fragmented cRNA to hybridize to the chip. If you start with 10 ug, there is a very high probability that you will end up with at least 16 ug at the end of the molecular biology step. However, if you only start with 8 ug, you are less likely to end up with the magical 16 ug, but it is still highly probable. We recently did something like 8 chips with only ~5 ug for each, and had 16 ug at the end for all of them. 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 >>> Wiesner Vos <vos@stats.ox.ac.uk> 10/06/03 10:02AM >>> Thanks for the replies Min-han and Jim. I suspected that this may be the case (keeping the papers in mind about comparing expression measures on the Dilution data eg. Cope et al.) I suspected that the use 8ug would be perhaps not make much of a difference if you use an expression measure like for example RMA that seems to be reasonably independent of the actual amount of RNA on a chip. Thanks again for your help. Wiesner J. Vos Department of Statistics University of Oxford 1 South Parks Road OX1 3TG United Kingdom On Mon, 6 Oct 2003, Tan, MinHan wrote: > - Perhaps it is an policy issue with your microarray core facility, but > the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug > total RNA. > > - I routinely use 5 ug total RNA with no apparent problems. > > - So yes, you should be able to do single sample hybridizations. > > Regards, > Min-Han > > > > -----Original Message----- > From: Wiesner Vos [mailto:vos@stats.ox.ac.uk] > Sent: Monday, October 06, 2003 9:27 AM > To: bioconductor@stat.math.ethz.ch > Subject: [BioC] Pooling in microarray studies > > > > I have question arising to the pooling of mRNA > samples. Someone approached me about the > following problem: > > The study wants to use Affymetrix chips to study > changes in expression between a group of treated > mice and a group untreated mice. There are 10 mice > in each group. It is only possible to extract > 8 ug of RNA from each mouse, not enough for one chip. (According to the > experimenters they require 10 ug per > chip) So it is not possible to use biological > replicate chips for each individual mice. Now the issue > is whether to perhaps pool the RNA in each group > and carry out analysis on technical replicates from the pooled > samples. > > As I understand it pooling may reduce the precision, with > the risk that one or few samples can dominate the outcome, and that > averaging over single sample hybridisations is perhaps safer than using > pooled samples. However in this case you cannot do single sample > hybridisations. > > I was wondering if the following approach is an acceptable compromise to > retain at least some information on the between sample variation in each > group: > > Mix the RNA from 2 different mice on a single chip to get 5 > hybridisations, where the hybridisation on each chip is from the mix of > the RNA samples of two mice? I though that this may enable you to some > extend if all the mice are behaving similarly. Ofcourse one would not be > able to distinguish between the behaviour of the two mice relating to > the same chip. Or is it better to accept that you do not have enough RNA > to hybridize the sample for each individual to a separate chip and pool > the samples and accept the risk that one sample may dominate the > outcome? The best solution did not seem obvious (to me at least!) > > Any comments will be much appreciated. > > Wiesner > > > > Wiesner J. Vos > Department of Statistics > University of Oxford > 1 South Parks Road > OX1 3TG > United Kingdom > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. > > _______________________________________________ > 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 _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you.
ADD COMMENTlink written 16.1 years ago by Tan, MinHan180
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