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Microarray Analysis – Analytical Techniques in Biotechnology

by Kevin Ahern, PhD
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    00:00 Now, the analytical tool I wanna talk about here, is actually used in the discipline of transcriptomics and it's called a microarray analysis.

    00:09 Now, microarray analysis is an amazingly powerful technique that allows a researcher to measure how much of every given messenger RNA is being made, okay? Now, microarray, in general, can analyze DNA and analyze protein. It can analyze RNA and other things.

    00:26 But this analysis of transcriptomics that I am going to talk about relates to messenger RNAs being made in a cell.

    00:34 How does it work? Well, you see in front of you a grid and that grid has little spots on it and those little spots are targets for molecules we are going to make and attach there, okay? So, we can see here that the grid is now being defined a little bit more with color.

    00:52 We know for example the sequence of the human genome.

    00:54 We know every base in it. We know every gene in it.

    00:58 And we would like to understand well how much of every gene in the human genome is being made in this particular cell type, let's say muscle.

    01:08 Well what would I do? I would take the sequence that I know and I would go to a chemist and I would have the chemist chemically synthesize and yes that's possible to do, chemically synthesize the sequence of each gene.

    01:25 So let's say I have a gene for hemoglobin as an example and I take that sequence for hemoglobin and I tell the chemist "Make me a few thousand copies of that sequence of that gene." And then take the sequence of another gene let's say hexokinase and make a few thousands copies of that.

    01:45 Now I am keeping each one of those separate.

    01:48 So the hemoglobin I might take for example and chemically bond it to that first upper left square that you see on the grid.

    01:57 I might take the hexokinase and link it to the other.

    02:00 It doesn't really matter what order I put them in as long as I keep track of which gene is in each place.

    02:06 So gene 1 will have thousands of copies of the DNA Gene 2 will have thousands of copies of a different DNA and gene 3 will have etc. We could do this on a microarray for every gene of 30,000 genes that human cells make, okay? This is a power miniaturization.

    02:28 What we do with that then, is we now have a grid that contains the sequence of every gene in the human genome.

    02:37 We wanna use this to study, let's say cancer.

    02:40 So, we take a healthy cell so let's again say we have got muscles.

    02:45 We take a healthy set of muscle cells.

    02:48 We isolate the messenger RNAs from that and then we have got a cancer that came from a muscle cell and we isolate all the messenger RNAs from that. We are keeping them separate at the moment.

    03:01 So there we have a copy of all the messenger RNAs of each cell type, normal and cancerous.

    03:10 We then copy each messenger RNA using reverse transcriptase.

    03:13 Now, reverse transcriptase I talked about in another presentation but it's an enzyme that copies RNA and makes DNA from it.

    03:24 Now the reason for using DNA is, it's a little easier to handle DNA than it is to handle messenger RNA so that's the reason that we do it.

    03:32 We then take those cDNAs, a cDNA is simply means its a copy of the RNA.

    03:38 We take the cDNAs and we add fluorescent tags to them.

    03:44 So for all the cDNAs that are in the healthy cells put a green tag on it. It's simply a chemical.

    03:51 On the cancer cells we taken we add a red tag to it.

    03:55 So we have two batches of messenger RNA now that have been flagged with each one with an appropriate color.

    04:02 Then we do something surprising. We take these samples that we have been so careful to keep apart and we mix them.

    04:10 And the reason that we mix them is we wanna give each one of these an equal opportunity to mix on this plate.

    04:18 So we take them and we pour the sample onto the plate that now has every possible gene on it.

    04:23 We allow hybridization to occur and that simply means finding the base pairs of each other like the primers of DNA found the strands in the PCR process.

    04:33 We allow hybridization to occur such that only strands that pair properly will pair with each other.

    04:41 So if there are for example messenger RNAs in there for hemoglobin they are gonna go to the spot where the hemoglobin is and anneal with it.

    04:48 Okay.

    04:50 We then take the plate and wash away anything that hasn't hybridized; because, things that haven't hybridized we are not really interested in.

    04:58 And then we take the plate to a plate reader and we analyze what's there.

    05:02 Now remember that the healthy cells had a green fluorescent tag and the cancers sells had a red fluorescent tag.

    05:11 This is what a microarray analysis might look like.

    05:14 The intensity of the color of the spot that we see on here, first of all, is a measure of the quantity of messenger RNA that was in each cell.

    05:23 The shade of the color tells us the relative expression of that messenger RNA in each cell type.

    05:30 So let's see what that means. Bright green means for example bright what indicate, it's abundant and green would indicate it's in healthy cells but it's not in cancers cells.

    05:44 Bright red would mean a gene that is abundant in cancer cells but not in healthy cells.

    05:51 Bright yellow means it's abundant in both cell types; because, in this kind of analysis when you mix green and red, believe it or not, you get yellow.

    06:01 Black would means it's absent in both cell types; because, not every gene is made inside of every cell.

    06:06 Now the beauty of this analysis is you can very quickly on one plate not only see what genes are being made but you can see where the differences are between two different cell types.

    06:20 You can understand that why is that protein being made in a cancer cell and not a regular cell, maybe that's a protein that I can design a drug for and knock out a cancer cell.

    06:33 So this technique is incredibly powerful that allowing us to use this information to design therapies and better understand how it is the cell's function.


    About the Lecture

    The lecture Microarray Analysis – Analytical Techniques in Biotechnology by Kevin Ahern, PhD is from the course Analytical Techniques in Biotechnology.


    Included Quiz Questions

    1. It can be performed with microarray analysis
    2. It tells how much protein is in a cell
    3. It focuses mostly on rRNA
    4. It focuses mostly on tRNA
    1. RNA fragments are attached to a grid
    2. DNA fragments are attached to a grid
    3. Annealing of RNAs to the DNA is the key step in the process
    4. mRNAs are the RNAs being analyzed
    1. Determine the gene expressions and differentiate between cell types simultaneously on the single plate
    2. Determine the gene expressions and differentiate between cell types at the same time on the two different plates having different target spots on them
    3. Understand the relationship between DNA and RNA inside a dying cell
    4. Estimates the relative amounts of protein to DNA in a cell growing under stressed conditions
    5. Estimate the relative amounts of protein to RNA in a cell under normal or standard conditions
    1. Bright blue color — mRNA expressed in both normal and cancer cells
    2. Bright green color — mRNA expressed in normal cells
    3. Bright red color — mRNA expressed in cancer cells
    4. Black color — mRNA expression absent in both normal and cancer cells
    5. Bright yellow color — mRNA expressed in both normal and cancer cells
    1. Quantity and relative expression of the expressed transcriptome in the samples, respectively
    2. Quantity and relative expression of rRNA in the samples, respectively
    3. Quantity and relative expression of tRNA in the samples, respectively
    4. Relative expression and quantity of expressed mRNA in the samples, respectively
    5. Relative quantities of the cell membrane and nuclear membranes in the samples

    Author of lecture Microarray Analysis – Analytical Techniques in Biotechnology

     Kevin Ahern, PhD

    Kevin Ahern, PhD


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