Different expression direction between limma microarray data analysis vs quantitative real time PCR result
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Wang, Jixin ▴ 90
@wang-jixin-3327
Last seen 9.6 years ago
Dear all, I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. Best regards, Wang
Microarray limma Microarray limma • 1.8k views
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Naomi Altman ★ 6.0k
@naomi-altman-380
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False discoveries? --Naomi At 12:38 AM 9/21/2009, jixinwang at tamu.edu wrote: >Dear all, I use limma package to do microarray >data analysis and most of real time PCR >validation results are consistent with >microarray data in terms of expression direction >and statistical significance. However in one set >of experiemnts, two DE genes are statistically >significant in both of microarray and qRT-PCR >result BUT had the different expression >direction. (e.g. They are down regulated in >microarray result but real time PCR result >showed that they are up regulated)? I don???t >know why. Does this occur to anyone before? Many >thanks. Best regards, Wang >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111
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Chao-Jen Wong ▴ 580
@chao-jen-wong-3603
Last seen 9.3 years ago
USA/Seattle/Fred Hutchinson Cancer Rese…
Hi, Jixin, Is your qRT-PCR expression level represented by cycle number (Ct) for limma analysis? If it is, then one thing I can think of is that you need to interpret the results in the opposite way. Since lower Ct means higher expression level, the resulting negative t or B values indicate up-regulation of the genes, not down-regulation. If I am wrong, please correct me. Regards, Chao-Jen jixinwang at tamu.edu wrote: > Dear all, > > I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. > > > Best regards, > > Wang > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- Chao-Jen Wong Program in Computational Biology Division of Public Health Sciences Fred Hutchinson Cancer Research Center 1100 Fairview Avenue N., M2-B876 PO Box 19024 Seattle, WA 98109 206.667.4485 cwon2 at fhcrc.org
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Hi, Chao-Jen, Thanks for kind reply. I use limma for microarray data analysis. But for qRT-PCR, I first selected the optimal number of internal control genes by geNorm program, and then use those housekeeping genes that received best score for normalization of qRT-PCR. The qBasePlus software (Biogazelle, Belgium) was used to evaluate the relative gene expression across tissues and the statistical significance of the derived CNRQ values was determined by SPSS 17.0 statistics software. So I don?t think either microarray or real time analysis has problem. Best regards, Wang ----- Original Message ----- From: "Chao-Jen Wong" <cwon2@fhcrc.org> To: jixinwang at tamu.edu Cc: bioconductor at stat.math.ethz.ch Sent: Monday, September 21, 2009 12:29:51 PM GMT -06:00 US/Canada Central Subject: Re: [BioC] Different expression direction between limma microarray data analysis vs quantitative real time PCR result Hi, Jixin, Is your qRT-PCR expression level represented by cycle number (Ct) for limma analysis? If it is, then one thing I can think of is that you need to interpret the results in the opposite way. Since lower Ct means higher expression level, the resulting negative t or B values indicate up-regulation of the genes, not down-regulation. If I am wrong, please correct me. Regards, Chao-Jen jixinwang at tamu.edu wrote: > Dear all, > > I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. > > > Best regards, > > Wang > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- Chao-Jen Wong Program in Computational Biology Division of Public Health Sciences Fred Hutchinson Cancer Research Center 1100 Fairview Avenue N., M2-B876 PO Box 19024 Seattle, WA 98109 206.667.4485 cwon2 at fhcrc.org
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Hi Wang, One more alternative: it's possible that both the microarray data and the qRT-PCR are "correct", if the microarray probe and the PCR primers target different parts of the transcript. There could be a splice variant (or something) between your conditions, such that one part of the transcript is higher in one condition but another part of the transcript is higher in the other condition. I saw a similar situation once with a KO gene where they removed a couple exons, and PCR primers based on those exons showed little or no transcript, but the microarray probes were for a different exon that was actually over-expressed, because there was no functional protein and the cell was trying to increase transcription! I believe the MACQ project also tried to track down the cause of some genes having different results on different microarray platforms and/or qRT-PCR results, and in almost all of the cases the probes were for very different parts of the transcript. Cheers, Jenny At 06:50 PM 9/21/2009, Wang, Jixin wrote: >Hi, Chao-Jen, Thanks for kind reply. I use limma >for microarray data analysis. But for qRT-PCR, I >first selected the optimal number of internal >control genes by geNorm program, and then use >those housekeeping genes that received best >score for normalization of qRT-PCR. The >qBasePlus software (Biogazelle, Belgium) was >used to evaluate the relative gene expression >across tissues and the statistical significance >of the derived CNRQ values was determined by >SPSS 17.0 statistics software. So I don???t >think either microarray or real time analysis >has problem. Best regards, Wang ----- Original >Message ----- From: "Chao-Jen Wong" ><cwon2 at="" fhcrc.org=""> To: jixinwang at tamu.edu Cc: >bioconductor at stat.math.ethz.ch Sent: Monday, >September 21, 2009 12:29:51 PM GMT -06:00 >US/Canada Central Subject: Re: [BioC] Different >expression direction between limma microarray >data analysis vs quantitative real time PCR >result Hi, Jixin, Is your qRT-PCR expression >level represented by cycle number (Ct) for limma >analysis? If it is, then one thing I can think >of is that you need to interpret the results in >the opposite way. Since lower Ct means higher >expression level, the resulting negative t or B >values indicate up-regulation of the genes, not >down-regulation. If I am wrong, please correct >me. Regards, Chao-Jen jixinwang at tamu.edu >wrote: > Dear all, > > I use limma package to do >microarray data analysis and most of real time >PCR validation results are consistent with >microarray data in terms of expression direction >and statistical significance. However in one set >of experiemnts, two DE genes are statistically >significant in both of microarray and qRT-PCR >result BUT had the different expression >direction. (e.g. They are down regulated in >microarray result but real time PCR result >showed that they are up regulated)? I don???t >know why. Does this occur to anyone before? Many >thanks. > > > Best regards, > > Wang > > >_______________________________________________ > > Bioconductor mailing list > >Bioconductor at stat.math.ethz.ch > >https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor >-- Chao-Jen Wong Program in Computational >Biology Division of Public Health Sciences Fred >Hutchinson Cancer Research Center 1100 Fairview >Avenue N., M2-B876 PO Box 19024 Seattle, WA >98109 206.667.4485 cwon2 at fhcrc.org >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor Jenny Drnevich, Ph.D. Functional Genomics Bioinformatics Specialist W.M. Keck Center for Comparative and Functional Genomics Roy J. Carver Biotechnology Center University of Illinois, Urbana-Champaign 330 ERML 1201 W. Gregory Dr. Urbana, IL 61801 USA ph: 217-244-7355 fax: 217-265-5066 e-mail: drnevich at illinois.edu
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Hi Jenny, Thanks so much for the kind help! I will check the probe and PCR product sequence to the genome and see what is going on. Best regards, Wang ----- Original Message ----- From: "Jenny Drnevich" <drnevich@illinois.edu> To: "Jixin Wang" <jixinwang at="" tamu.edu="">, "Chao-Jen Wong" <cwon2 at="" fhcrc.org=""> Cc: bioconductor at stat.math.ethz.ch Sent: Tuesday, September 22, 2009 8:54:13 AM GMT -06:00 US/Canada Central Subject: Re: [BioC] Different expression direction between limma microarray data analysis vs quantitative real time PCR result Hi Wang, One more alternative: it's possible that both the microarray data and the qRT-PCR are "correct", if the microarray probe and the PCR primers target different parts of the transcript. There could be a splice variant (or something) between your conditions, such that one part of the transcript is higher in one condition but another part of the transcript is higher in the other condition. I saw a similar situation once with a KO gene where they removed a couple exons, and PCR primers based on those exons showed little or no transcript, but the microarray probes were for a different exon that was actually over-expressed, because there was no functional protein and the cell was trying to increase transcription! I believe the MACQ project also tried to track down the cause of some genes having different results on different microarray platforms and/or qRT-PCR results, and in almost all of the cases the probes were for very different parts of the transcript. Cheers, Jenny At 06:50 PM 9/21/2009, Wang, Jixin wrote: >Hi, Chao-Jen, Thanks for kind reply. I use limma >for microarray data analysis. But for qRT-PCR, I >first selected the optimal number of internal >control genes by geNorm program, and then use >those housekeeping genes that received best >score for normalization of qRT-PCR. The >qBasePlus software (Biogazelle, Belgium) was >used to evaluate the relative gene expression >across tissues and the statistical significance >of the derived CNRQ values was determined by >SPSS 17.0 statistics software. So I don???t >think either microarray or real time analysis >has problem. Best regards, Wang ----- Original >Message ----- From: "Chao-Jen Wong" ><cwon2 at="" fhcrc.org=""> To: jixinwang at tamu.edu Cc: >bioconductor at stat.math.ethz.ch Sent: Monday, >September 21, 2009 12:29:51 PM GMT -06:00 >US/Canada Central Subject: Re: [BioC] Different >expression direction between limma microarray >data analysis vs quantitative real time PCR >result Hi, Jixin, Is your qRT-PCR expression >level represented by cycle number (Ct) for limma >analysis? If it is, then one thing I can think >of is that you need to interpret the results in >the opposite way. Since lower Ct means higher >expression level, the resulting negative t or B >values indicate up-regulation of the genes, not >down-regulation. If I am wrong, please correct >me. Regards, Chao-Jen jixinwang at tamu.edu >wrote: > Dear all, > > I use limma package to do >microarray data analysis and most of real time >PCR validation results are consistent with >microarray data in terms of expression direction >and statistical significance. However in one set >of experiemnts, two DE genes are statistically >significant in both of microarray and qRT-PCR >result BUT had the different expression >direction. (e.g. They are down regulated in >microarray result but real time PCR result >showed that they are up regulated)? I don???t >know why. Does this occur to anyone before? Many >thanks. > > > Best regards, > > Wang > > >_______________________________________________ > > Bioconductor mailing list > >Bioconductor at stat.math.ethz.ch > >https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor >-- Chao-Jen Wong Program in Computational >Biology Division of Public Health Sciences Fred >Hutchinson Cancer Research Center 1100 Fairview >Avenue N., M2-B876 PO Box 19024 Seattle, WA >98109 206.667.4485 cwon2 at fhcrc.org >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor Jenny Drnevich, Ph.D. Functional Genomics Bioinformatics Specialist W.M. Keck Center for Comparative and Functional Genomics Roy J. Carver Biotechnology Center University of Illinois, Urbana-Champaign 330 ERML 1201 W. Gregory Dr. Urbana, IL 61801 USA ph: 217-244-7355 fax: 217-265-5066 e-mail: drnevich at illinois.edu
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Francois Pepin ★ 1.3k
@francois-pepin-1012
Last seen 9.6 years ago
Hi Wang, Is it 2 genes out of say 10 (or more), or two genes out of two? If it's two out of 10 and the others worked out properly, I would go with Naomi and say it's probably just a false discovery. If you only checked two, the first thing that comes to mind is that you had the samples switched at some point, either on the microarray side or the PCR side. In this case, you might want to do a few more genes to see what's happening. Francois jixinwang at tamu.edu wrote: > Dear all, > > I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. > > > Best regards, > > Wang > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
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Hi Francois and Naomi, Thanks so much for the help. It is 2 genes out of eleven. I had checked the samples used for both of microarray and real time PCR. Best Regards, Wang ----- Original Message ----- From: "Francois Pepin" <fpepin@cs.mcgill.ca> To: jixinwang at tamu.edu Cc: bioconductor at stat.math.ethz.ch Sent: Monday, September 21, 2009 9:31:36 AM GMT -06:00 US/Canada Central Subject: Re: [BioC] Different expression direction between limma microarray data analysis vs quantitative real time PCR result Hi Wang, Is it 2 genes out of say 10 (or more), or two genes out of two? If it's two out of 10 and the others worked out properly, I would go with Naomi and say it's probably just a false discovery. If you only checked two, the first thing that comes to mind is that you had the samples switched at some point, either on the microarray side or the PCR side. In this case, you might want to do a few more genes to see what's happening. Francois jixinwang at tamu.edu wrote: > Dear all, > > I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. > > > Best regards, > > Wang > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
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De-Jian ZHAO ▴ 240
@de-jian-zhao-2012
Last seen 9.6 years ago
Have you ever checked the original microarray images? Sometimes some badly-printed spots may pass the QC and lead to this kind of weired problems. Chances are higher if you have less replicates. I'm just fancying.... jixinwang at tamu.edu wrote: > Dear all, > > I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and qRT-PCR result BUT had the different expression direction. (e.g. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? I don?t know why. Does this occur to anyone before? Many thanks. > > > Best regards, > > Wang > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
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