FEATURE ARTICLE
Gene Chips and Functional Genomics
A new technology will allow environmental health scientists to track the expression of thousands of genes in a single, fast and easy test
Hisham Hamadeh, Cynthia Afshari
Comparing Samples
Once the gene chip is prepared and is spotted with the entire set of cDNAs from the cell or tissue of interest, investigators can use it to look at gene expression in various cells and organs under different circumstances. This again is done by withdrawing message molecules from the sample cells or organs to see exactly which subset of genes is being expressed at any given moment. In this way, patterns of gene expression may be compared from a normal versus diseased tissue, an untreated versus drug or chemically treated cell, or a normal versus a mutant cell.
Messages in the form of RNA may be derived from any species, including bacteria, fungi, plants or animals. Most RNA molecules code for protein and are shuttled from the cell nucleus to the cytoplasm and there are translated into protein. These so-called messenger RNA molecules (mRNA) can contain between 400 to 10,000 nucleotides, the sequence of which serves to indicate the corresponding amino acid sequence of a protein.
The number of copies of the RNA "message" transcribed from a gene often relates directly to the quantity of protein ultimately produced. For example, if a large amount of protein will be required, a large number of RNA transcripts for that protein will probably be present in the cell's cytoplasm. A small amount of RNA in the cytoplasm may indicate that a small amount of protein is to be made. Therefore, by measuring the amount of message in the cell, scientists can not only determine which genes are being expressed, they can also determine at what levels they are being expressed. This is especially useful for comparisons between samples. Often scientists want to know whether certain conditions alter the level of gene expression—that is, whether a particular gene is expressed in greater or lesser quantities in response to some environmental exposure.
Differential expression measurements are carried out using a simultaneous, two-color fluorescence hybridization scheme. cDNAs are converted from RNA molecules in the presence of nucleotides to which a fluorescent colored dye has been attached. Different colors can be given to the message derived from different cells, such that the message from the control cells can be tagged green, for example, whereas the message from chemically treated cells might be tagged red.
The color-coding allows for a very clear way of comparing the quantities of message from each cell. The fluorescently labeled messages derived from different groups of cells are mixed and bound simultaneously on the same cDNA microarray chip. The array is then optically scanned at two wavelengths using independent lasers to excite the two fluorescent dyes at 632 and 532 nanometers for the red and green labels, respectively. Information from the scanner is translated into images corresponding to the two dyes scanned, and this is sent to a computer for further analysis. The computer produces two different pictures of the gene chip. One shows the location of all the spots lit in green and notes the intensity of the green spots. The other picture notes the location and intensity of all of the red spots.
These two images are then overlaid using specialized software that assigns a color for each tag and compares the two images. The result is a very colorful display of spots ranging between green and red. From this, scientists can determine the relative abundance of message manufactured in each cell.
For example, if a gene is expressed in equal amounts in both the control and the treated samples, then the computer will note equal amounts of red dye and green dye. The computer will indicate such a spot as yellow. Should a gene be highly expressed in the control cell, the one where the messages are colored green, but less so in the treated cell, then the spot will show up as predominantly green. If the gene is expressed in the treated cell but less so in the control, the spot will show up as predominantly red.
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