Dear Bioc, Has someone ever used long oligo arrays to test if genes were expressed or not? I have two microarray slides on which oligonucleotides (65mer) for genes from a parasite genome were spotted. One gene has one oligo but spotted twice on each array. One slide has a RNA sample extracted from infected tissues (therefore mixture of both parasite RNAs and RNAs from host tissues) co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). Another is a biological replicate but with dye-swap. People want to know what parasite genes are expressed in the infected tissue. Is it possible to do so? Related publications are highly appreciated. Best, Xinxia

On 1/13/06 3:07 AM, "xpeng" <xpeng at="" utk.edu=""> wrote: > Dear Bioc, > > Has someone ever used long oligo arrays to test if genes were expressed or > not? I have two microarray slides on which oligonucleotides (65mer) for genes > from a parasite genome were spotted. One gene has one oligo but spotted twice > on each array. One slide has a RNA sample extracted from infected tissues > (therefore mixture of both parasite RNAs and RNAs from host tissues) > co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). > Another is a biological replicate but with dye-swap. People want to know what > parasite genes are expressed in the infected tissue. Is it possible to do so? > Related publications are highly appreciated. You cannot answer that DIRECTLY with this design, I don't think. You could look for differential expression between the two samples--that is probably as close as you can get. You can perhaps say what genes are NOT expressed, as those spots will have very low intensity (within the range of background), but that doesn't imply that the others are expressed. And, you might think that all genes that show some signal AND are differentially expressed between the two samples would be expressed parasite genes, but these could also be due to regulation of the genes in the infected host combined with some cross-hybridization. So, I would look for differentially-expressed genes and see what that can tell you. With only two samples, doing meaningful statistics will be difficult, but you could try using limma or some other moderated testing procedure designed for small samples (and able to account for your replicated probes). Alternatively, just rank the genes by fold change. In the end, if you REALLY want to know which genes are differentially expressed, another couple of arrays would probably help. Sean

Hi Amy, Thanks for the reply. You are right. The uninfected tissue is a blank, in theory. But the real problem is that some probes cross-hybridize with house RNAs. I do not know the oligo sequence yet. They know that there are more host RNAs than parasite RNAs in the sample from the infected tissue, but not sure the percentage. Yes, they are 2-color arrays. The problem here is what to compare. It was suggested to find genes with intensities higher than their own backgrounds, say 4-fold higer, but I doubt it. I appreciate more inputs on the consequences if it is done this way. Best, Xinxia >===== Original Message From a.mikhail at abdn.ac.uk ===== >Hi Xinxia, > >Just curious about your message. It is quite common to look at parasite >genes being expressed at different stages of the parasite lifecycle, >including when the parasite is in the host, as is the case for your >infected tissue. However I'm a bit confused about the RNA you >cohybridised with, as if there are no parasite RNAs in it you would be >comparing parasite gene expression in your infected tissue with a blank >(did I read correctly that your microarrays have only parasite genes on >them, not host genes?). > >Are you using 2-colour arrays? If so expression levels for each gene are >normally read from one condition relative to the other. This means that >whatever you co-hybridised with has to have the same set of genes being >expressed at some level - otherwise you have nothing to compare your >condition of interest with. The results you get in this way are relative >expression levels for each gene of interest, not absolute expression >levels. As I understand it you cannot use microarrays for absolute >expression levels because there are too many factors that can disrupt mRNA >copy number during probe preparation - if you are interested in actual >copy number you would have to do something like quantitative RT-PCR. > >Hope this helps, > >Regards, >Amy > >--------------------------------------------------------------------- ------- >Dear Bioc, > >Has someone ever used long oligo arrays to test if genes were expressed or > >not? I have two microarray slides on which oligonucleotides (65mer) for genes >from a parasite genome were spotted. One gene has one oligo but spotted twice >on each array. One slide has a RNA sample extracted from infected tissues >(therefore mixture of both parasite RNAs and RNAs from host tissues) >co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). >Another is a biological replicate but with dye-swap. People want to know >what >parasite genes are expressed in the infected tissue. Is it possible to do so? >Related publications are highly appreciated. > >Best, >Xinxia >--------------------------------------------------------------------- ------- > > > >------------------------------------------- >Amy Mikhail >Research student >University of Aberdeen >Zoology Building >Tillydrone Avenue >Aberdeen AB24 2TZ >Scotland >Email: a.mikhail at abdn.ac.uk >Phone: 00-44-1224-272880 (lab)

I agree. Thanks a lot. Even I want to find differentially expressed genes. How do I normalize? The oligos are designed based on parasite genes, while samples from uninfected tissue have no parasite RNAs. Best, Xinxia >===== Original Message From Sean Davis <sdavis2 at="" mail.nih.gov=""> ===== >On 1/13/06 3:07 AM, "xpeng" <xpeng at="" utk.edu=""> wrote: > >> Dear Bioc, >> >> Has someone ever used long oligo arrays to test if genes were expressed or >> not? I have two microarray slides on which oligonucleotides (65mer) for genes >> from a parasite genome were spotted. One gene has one oligo but spotted twice >> on each array. One slide has a RNA sample extracted from infected tissues >> (therefore mixture of both parasite RNAs and RNAs from host tissues) >> co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). >> Another is a biological replicate but with dye-swap. People want to know what >> parasite genes are expressed in the infected tissue. Is it possible to do so? >> Related publications are highly appreciated. > >You cannot answer that DIRECTLY with this design, I don't think. You could >look for differential expression between the two samples--that is probably >as close as you can get. You can perhaps say what genes are NOT expressed, >as those spots will have very low intensity (within the range of >background), but that doesn't imply that the others are expressed. And, you >might think that all genes that show some signal AND are differentially >expressed between the two samples would be expressed parasite genes, but >these could also be due to regulation of the genes in the infected host >combined with some cross-hybridization. So, I would look for >differentially-expressed genes and see what that can tell you. With only >two samples, doing meaningful statistics will be difficult, but you could >try using limma or some other moderated testing procedure designed for small >samples (and able to account for your replicated probes). Alternatively, >just rank the genes by fold change. In the end, if you REALLY want to know >which genes are differentially expressed, another couple of arrays would >probably help. > >Sean > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor

Thanks a lot. What are the arrays or other technologies to survey the presence/absence of genes in a large scale? I noticed that Winzeler's lab published a paper using a Affy-like custom-made, high-density oligo array (Le Roch et al, Science, 301:1503-1508). Best, Xinxia >===== Original Message From Sean Davis <sdavis2 at="" mail.nih.gov=""> ===== >On 1/13/06 12:49 PM, "xpeng" <xpeng at="" utk.edu=""> wrote: > >> Hi Amy, >> >> Thanks for the reply. >> >> You are right. The uninfected tissue is a blank, in theory. But the real >> problem is that some probes cross-hybridize with house RNAs. I do not know the >> oligo sequence yet. They know that there are more host RNAs than parasite RNAs >> in the sample from the infected tissue, but not sure the percentage. >> >> Yes, they are 2-color arrays. The problem here is what to compare. It was >> suggested to find genes with intensities higher than their own backgrounds, >> say 4-fold higer, but I doubt it. I appreciate more inputs on the consequences >> if it is done this way. > >Signals above background in the infected sample could be due to at least >three possibilities: > >1) Hybridization by the parasite RNA >2) Cross-hyb by the host RNA in the uninfected state >3) Additional cross-hyb by host RNA due to the infection (upregulation of >genes not highly expressed in the uninfected state). > >I agree with Amy that the best you can probably do is to look for >differentially-expressed genes between your uninfected host and the infected >sample. I also agree with Amy that using absolute intensities as a measure >of "expression" is probably not possible with most existing long- oligo >technologies (although some new technologies like illumina seem to be >working somewhat for that). > >Sean

Hi Xinxia, Just curious about your message. It is quite common to look at parasite genes being expressed at different stages of the parasite lifecycle, including when the parasite is in the host, as is the case for your infected tissue. However I'm a bit confused about the RNA you cohybridised with, as if there are no parasite RNAs in it you would be comparing parasite gene expression in your infected tissue with a blank (did I read correctly that your microarrays have only parasite genes on them, not host genes?). Are you using 2-colour arrays? If so expression levels for each gene are normally read from one condition relative to the other. This means that whatever you co-hybridised with has to have the same set of genes being expressed at some level - otherwise you have nothing to compare your condition of interest with. The results you get in this way are relative expression levels for each gene of interest, not absolute expression levels. As I understand it you cannot use microarrays for absolute expression levels because there are too many factors that can disrupt mRNA copy number during probe preparation - if you are interested in actual copy number you would have to do something like quantitative RT-PCR. Hope this helps, Regards, Amy ---------------------------------------------------------------------- ------ Dear Bioc, Has someone ever used long oligo arrays to test if genes were expressed or not? I have two microarray slides on which oligonucleotides (65mer) for genes from a parasite genome were spotted. One gene has one oligo but spotted twice on each array. One slide has a RNA sample extracted from infected tissues (therefore mixture of both parasite RNAs and RNAs from host tissues) co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). Another is a biological replicate but with dye-swap. People want to know what parasite genes are expressed in the infected tissue. Is it possible to do so? Related publications are highly appreciated. Best, Xinxia ---------------------------------------------------------------------- ------ ------------------------------------------- Amy Mikhail Research student University of Aberdeen Zoology Building Tillydrone Avenue Aberdeen AB24 2TZ Scotland Email: a.mikhail at abdn.ac.uk Phone: 00-44-1224-272880 (lab)