Linda Wolin - Microarrays

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Microarrays

By Linda Wolin
President Writesaif, Inc.
www.writesaif.com
writesaif@sbcglobal.net

On June 26th, 2000(1), President Clinton announced with Dr. Francis Collins (Director of the international Human Genome Project) (1) and Dr. Craig Venter (President and CSO, Celera Genomics) (1) that the first audit of the entire human genome was documented and viewable.(1,2) With this communication, a door opened for scientists to vast amounts of new information on human-DNA sequences.

Around the same time, a new class of tool was being recognized by researchers, a tool that enabled the collection and analysis of vast amounts of information, effectively and efficiently. This class of tool was the microarray, so scientists started using microarrays to analyze the new genomic information.

Relevant Scientific Facts

Each of us has a unique set of genes in the cells of our body that contain our inherited characteristics.(3) Our genes, however, do more then pass on characteristics; they also regulate protein formation.(3) The means by which they to this is very mysterious, complex and for the most part, still unkonwn.(2) Scientists are just beginning to understand the processes used. What is known is that genes do not and can not work all of the time! They only work when "turned on" or "expressed," similar to a car, which works only when its engine is started. Once genes are "expressed," information stored in them gets transcribed or transferred "from one recording or storing system to another."(2,4) The information moves from our DNA to some of our RNA, and the RNA becomes the new storage system. The portion of our RNA that receives the information becomes known as "messenger RNA" or mRNA.(2,4,5) Our mRNA then translates (or changes to another form or language) our genetic information into proteins.(2,4,5) If our genetic information is abnormally changed, when mRNA translates, our proteins' structures and functions change, which may lead to disease, advancement of existing disease, or birth defects.(4)

Parts of Microarrays

Today, microarrays are manufactured by multiple companies (two of which are Affimetrix, and Agilent Technologies).(6) Manufacturing processes vary, but all microarrays have two-main parts.(4,7)

A support or substrate(4,7)
Identified genetic probes (4)

Each microarry has a support or substrate that is typically a glass slide, nylon membrane, or a silicon chip.(2,4,7) Placed on the substrate are identified pieces of a gene sequence. (If a microarray experiment is used to analyze human tissue, then the identified pieces are identical to parts of the human genome.)(4) The identified gene pieces are not just placed on the substrate; rather, they are attached, identified, and arranged in patterns. (13) Hundreds of thousands(4) of them may be attached on one array! Once the genetic pieces are attached, they are referrred to as genetic probes.(4)

Uses of Microarrays

One of three different types of genetic material may be used for a microarray's genetic probes, DNA, cDNA or mRNA.(4,8) The type used for the probes depends on what type of information is needed from a microarray experiment. DNA probes from a single gene are used to test an individual for the potential to develop a disease; cDNA probes are used if the stage of a disease needs to be clarified and mRNA probes are used for drug development, drug response, and therapy treatments.(8) More specifically, microarrays and related analyses of the information gained from them help provide answers to the following questions:

Which genes in an individual are "expressed" or "turned on"?(4)
How does diseased tissue differ from normal tissue?(4)
What stage of a disease is present in a person?(4)
Which drugs will be best for treatment?
How can drugs be improved for treatments? (4)
Which drugs work best at different stages of a disease?(4)
Which genes are acting together, as a cluster? (4)
What or which groups of genes are responsible for a hereditary characteristic, hereditary syndrome, or disease?(4,9)
What causes genes to be turned on or off?
Who is the individual who died whose identity is unknown? (10)
Who committed this crime?(10,11)

Microarray Experiments

A physician or researcher determines the type of information needed from a microarray experiment, and thus selects the type of genetic tissue (DNA, cDNA or mRNA) needed for study. This genetic tissue serves as the source for mobile probes that are applied to a manufactured microarray with immobilzed probes on it. The type of genetic tissue used (as the mobilized probes) is the same as the type that is already on the manufactured array. (For example, if the array has immobilized DNA pieces, then the mobile probes taken from tissue are composed of DNA also, not cDNA or mRNA.) The mobile probes may be applied by a solution process, which enables the mobilized and immobilized probes to be in contact with one another. After a designated period of time, some of the mobilized probes bind to some of the immobilized probes. The microarray is removed from the solution, scanned by a laser, and a digital image of the array is obtained. Data is stored and analyzed by a software program.(2, 4,7)

If a goal of the experiment is to compare two types of cells, such as cells of diseased and normal tissue, then the mobile probes may be given florescent tags,(2, 4,7) one color for the tissue from known-healthy cells, and another color for the tissue from diseased cells. Florescent tags, during analysis, show the different locations that healthy and diseased tissue bind to.

Experimental Errors

Accuracy in array analyses is extremely important due to the impact this information has on individuals whose tissue samples are being analyzed and the impact the information may have on future drug development and diagnostic and therapeutic assessments. However, errors in microarray analyses arise from differences caused by multiple factors some of which are:

Sample-tissue-cell variations.
Temperature differences of solutions and variances in solution-exposure times.
Varying expression levels of mRNA, which may differ by cell type.
Variations in the handling procedure for tissue samples (DNA, cDNA or nRNA).
Use of non-standard mark-up languages to identify attached probes on the array substrates.
Use of different scanning devices to document results and different statistical programs to analyze data.
Varitions in number, size, and distance between immobilized probes on the substrates. Similar variations may exist regarding the mobilized probes.
Use of different florescent levels in experiments.(7)
Background noise or confusion between probe, substrate or other particle characteristics, during scanning, which may lead to false positives or negatives.(7)

Recognized standards for the processes followed in microarray experiments are needed globally to reduce some of the errors that arise. The Microarray Gene Expression Database, MGED,(12) lists minimum information about a microarray experiment, MIAME,(12) that should be reported prior to journal submission.(13) The purposes of the standard are simliar to the need for standards with other scientific research: aid reproducibility of results and facilitate the interpretation of data and comparison of data between experiments. However, neither standard is recognized globally, to date.(13)

Future Trends

As use of microarray-technology advances, it is expected that the cost of arrays will decrease, due to supply-demand factors. (When customer demand increases, it becomes economically feasible to produce larger volumes, so unit costs for manufacture decrease. A recent example is provided by the digital camera, which initially was only available for hundreds of dollars. Now, some models can be purchased for slightly less than $100.00).

Other changes predicted for arrays include (1) The manufacture and use of simpler arrays (by physicians, in their offices) for quick and more-accurate-clinical diagnoses(14); (2) The acceptance of national (if not international) standards for array manufacture, scanning, and analysis; and (3) The emergence and increasing use of smaller nano-arrays, such as the ones presently manufactured by Bioforce Nanosciences. (On the latter, 1,500 different samples may be tested in the space presently needed for one sample on a microarray!)(15)

References

1. The White House, Office of the Press Secretary, Remarks by the President. June 26, 2000. Available at: link. Accessed: October 30, 2004.
2. Baldi, P., Hatfield G. W. DNA Microarrays and Gene Expression. From experiments to data analysis and modeling. Cambridge, United Kingdom: Cambridge University Press; 2002.
3. Moore J. From Genesis to Genetics. Berkeley, Los Angeles, London. University of California Press; 2002:121.
4. NCBI. Microarrays: Chipping away at the mysteries of science and medicine" Revised March 30, 2004. Available at: link. Accessed: October 13, 2004.
5. Forbes, N. Imitation of Lif., Cambridge, Mass. London, England. The MIT Press;2004;71.
6. Baldi, p.8.
7. Cheung et al. "Making and reading microarrays." Nature Genetics Supplement. Vol. 21 Jan., 1999. p. 15.
8. Baldi, p.13.
9. Nature Genetics, Assembly of microarrays for genome-wide measurement of DNA copy number." Vol 29. November 23, 2001. p. 2.
10. Williamson, R., Duncan, R. DNA testing for all. Nature, Vol.418 August 8, 2002. p. 585.
11. Hibbert, M. "DNA databanks: law enforcement's greatest surveillance tool." Wake Forest Law Review. 1999 Fall;34(3):767-825. Accessed: August 17, 2004.
12. MGED Society. MGED home page. Available at: link Microarray Gene Expression Data Society. Accessed October 30, 2004.
13. Nature Genetics. "Minimum information about a microarray experiment (MIAME)-toward standards for microarray data" Dec, 2001; 10.1038/ng1201-365 volume 29 no. 4 pp 365 - 371 Available at: link. Accessed 10/30/04.
14. Vaarno et al. New separation-free assay technique for SNPs using two-photon excitation flourometry. Nuclei Acids Research. Oxford University Press. 2004:Vol. 32 No13;1.
15. Bioforce Nanosciences, Inc. "Nanoarrays vs microarrays." 2004. Availble at: link. Accessed August 16, 2004.

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