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Why do we use two channels in MICRO ARRAY analysis ?

  1. May 10, 2004 #1
    Why do we use two channels in MICRO ARRAY analysis ??
    LIke red and Green ??
    What does the ratio between red/green signify ??
     
  2. jcsd
  3. May 10, 2004 #2

    Monique

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    It are two different samples on the same chip. You label one RNA sample with green fluorescent dye and the other sample with red fluorescent dye. You hybridize that to a chip, if the concentration of a particular RNA is the same in both samples, you'll get a yellow signal.. if one sample is stronger than the other you'll either get a red or a green signal. So the ratio is relative gene expression levels.
     
  4. May 10, 2004 #3

    Monique

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    You can make your own micro arrays, but there are also custom arrays available and even ones with every single gene (about 30000) of the human (or other) genome on it. Besides that they are working on making protein chips, with many different epitopes on them.
     
  5. May 11, 2004 #4
    Actually my work is on the analysis of the output.
    Now the problem is where can i get the output ?
    and in what format ? (intensity/ratio/ ... ) ???
    Any links ???
     
  6. May 11, 2004 #5

    Monique

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    Depends on the kind of array you are working on. There are house-keeping genes on chips that allow you to correct for the difference of total RNA of the two samples that went into the procedure originally, the expression level of the house-keeping genes should be the same.

    You probably have two samples that have a different biological source, like diseased vs healthy tissue. You'd be interesed in the ratio between the two samples, not absolute values.

    If you are looking for a biological marker for disease phenotype, it could be interesting though to specifically look for genes with high intensity and different ratio, since those are easier to detect. If you are looking for the biological foundation of a disease phenotype, it might be better worth looking at low expression genes. Those are more likely to be downstream of the signalling pathways and thus be the cause of other disease markers to be upregulated much more.

    What kind of chip and what kind of program are you working with?
     
  7. May 12, 2004 #6
    I just want the data file...

    Give me some URL to download ...
    No matter what kind of chip it is ...
     
  8. May 12, 2004 #7

    Monique

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    Good luck looking for it yourself.
     
  9. May 12, 2004 #8

    Moonbear

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    You're not going to find such a data file on the web. Anyone using the chips for research isn't going to put their raw data out on the web for just anyone to use. You might be able to contact the company that makes the software you're using to get a sample data set to give the software a "test drive", but all the software is copyrighted, and the data files probably are obtained in formats specific for the software bundled with the chip reader.
     
  10. May 13, 2004 #9

    Monique

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    Actually I think journals were planning to make available raw data from experiments published by them through the web (since published data should be free for everyone to look at).. look up a Nature article with microarrays and see if there is extra online content.
     
  11. May 13, 2004 #10
    Hey thanks guys.
    I found it in Stanford Microarray databse.

    Thanx for the those theories....

    BTW, Can we work with more than two genes at a time ?? I mean more than 2 dyes .. ??

    Coz, tumors may produce many genes...
    To identify them or to see the level of expresion of those genes....
     
  12. May 13, 2004 #11

    Monique

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    The two dyes are not genes, but samples. Affymetrix has human genome chips which carry all 30000 some genes, this method uses biotin labeled probes though and not fluorescent dyes.

    You could use three dyes, but there would be overlap in spectrum and you will get too much competition of the sample for binding to the probe (be careful, probe and target are often switched around in chip lingo, not sure myself which is which).
     
  13. May 14, 2004 #12
    So you mean .....
    Its Ideal to use only 2 samples and one gene in MICRO ARRAY ???

    or

    we compare only relative expression on of single gene in two samples ???


    So what does rows and columns in Micro Array represent ???
     
  14. May 14, 2004 #13

    Monique

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    Every spot on the microarray represents a single gene or transcript, they are short uniques pieces of DNA to which another piece can bind.

    For instance, you obtain biological samples of a patient and a control, you isolate RNA from that, you make cDNA and you label that with a dye. The patient cDNA you label red and the control you label green. You then hybridize this cDNA (which represents the gene pool that is being expressed in the sample) to the array with all the different pieces of genes spotted on it. You then wash away everything that doesn't bind. If a spot lights up, it means that in the expressed gene pool the particular transcript was present. If it lights up red, it means that transcript was expressed higher in the patient than in the control. If it lights up yellow, the expression levels were the same in both samples.
     
  15. May 14, 2004 #14

    Monique

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    There are all tricks on microarrays too, to determine the aspecifical binding and also the ratio of 3'/5' transcripts, not sure if you want to know..
     
  16. May 14, 2004 #15
    hey thanx.
    Got a doubt ...
    Do you label all the cDNAs from a sample ???

    If yes ! , how do you know which one(gene) glows ???

    Eg. THERE are N genes (probe) in slide. And u add P cDNAs to the slide. If u label all.
    How do we know whether its P1-N1 or P4-N4 ..... ????

    Im really confused day by day .
    PLease help me !!! :confused:

    The following Link confused me a lot
    http://genome-www5.stanford.edu/cgi-bin/data/grids.pl?fullID=21508GENEPIX21508
    its from Stanford Microarray Database.

    When we click on the spot on the image it shows the information about that.

    Different spots shows different genes.

    BTW,
    What are 5' and 3' transcripts ???
     
  17. May 14, 2004 #16

    Monique

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    An array is spotted by a robot, you have a platform with a lot of pins, each pin goes into a solution with a different DNA mix, a drop is transferred to the array slide. It is exactly known which spot is which DNA, there is software that places a grid over the spots and can read all the values of all the different spots and since it is an ordered matrix, gene names can be assigned to each spot.

    Spot A1 will always be ACE, spot A2 will always be TULP for example.

    So yes, you label ALL the cDNA (made from RNA) of a sample, depending whether the ACE gene spot lights up or the TULP gene spot, do you know which was in the sample and how much.

    Ofcourse it is possible that the TULP gene spot binds other cDNA than it is supposed to, aspecific binding. Commercial arrays measure this aspecific binding by including for instance 12 different probes for each gene. Some probes have mutations, if something still binds to it, it is aspecific and the signal should be subtracted from the signal of the original probe. But this all happens behind the scenes and you don't have to worry about it, but it is good to know.

    With the 5'/3' transcript ratio you measure the extend that your RNA template was degraded and how well the amplification procedure went. I think that if the amplification was not efficient, you will be losing the 5' end of the RNA. This is also important to know, since if the piece of DNA of the probe is in the 5' end, and you lost that during the sample preparation, you won't get a signal, while the 3' end of the molecule is still there. Commercial chips will use probes of the 5' end, 3' end, and the middle of an RNA to get an accurate reading (they are several out of those 12 probes I mentioned earlier).
     
  18. May 14, 2004 #17

    Monique

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    So: the DNA of the array is all synthetic and you know exactly what they represent. You throw on a mixture of two sample labeled with different colors. Depending on which spot lights up in which color, do you know what was in your sample
     
  19. May 20, 2004 #18

    Monique

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    Affymetrix - probe selection and array design
    It all depends on what you are doing, but generally when you amplify RNA you loose the 5' end of the transcript, that has to do with the way the primers anneal. For the 3' end you can make a primer with a poly-A tail, so you will always amplify the end. For the 5' end there is no common tail for all the different gene-transcripts, so a random primer is used. That primer can anneal anywhere in the transcript so you never know how full length your cDNA is. That is why there is a bias towards the 3', it depends how efficiently the amplification procedure went, whether there are long amplicons.

    So for instance if you're working with a custom array that was made and the probes are all from the 5' end of the target, there is a bias that signals can be missed. I'm showing that in order to analyze data, you need to know how they were generated in detail, how was the RNA amplified, how were the probes made, is the information acquired representive of the original material? .. science :)
     
  20. Jun 4, 2004 #19
    What are Predictors ????

    How do i find them in Micro array clusters ??

    How do i analyse the clusters formed by Micro Array data analysis ???
     
  21. Jun 4, 2004 #20

    Monique

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    Predictor: a reliable marker that allows you to give a prognosis or diagnosis on a patient for instance. You must know the answer to the other questions.
     
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