The analysis of DNA using the microarray technique has become one of the most significant methods in the area of research genetics. This technique falls under the area of gene expression profiling. Most of the time, this procedure is applied by scientists in the effort to investigate a wide range of conditions. This is because experimental procedures cam be performed on numerous genes at the same time. They include researches on cancer to finding numerous solutions to the problems that are presented by pests. With this advancement an opportunity has been offered for the performance of personal DNA microarray experiments. Among the basis of such experiments is the determination between healthy cells and the cancer cells. Based on the complexity of the microarray experiments, it is vital that all scientists obtain a solid understanding on the DNA basics as well as the way through which genes express themselves.
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DNA microarrays have been used in the extensive survey of the relative transcription in any gene within a genome. Most of the cancer cells in human beings are found within the developing nerves. Howevber, they do not allow the complete quantification level of gene expression. Moreover, the DNA chips do not make it possible to determine the amount of mRNA produced from a relative sample with that produced from the control population. As such, it can be used to compare the rate of gene expression in a lung cell with cancer and a helthy lung cell. Therefore, the main goal of this practival test is to ptovide a way to understand how microarrays are used tostudy the gene expressions. It allows the investigators to determine the level of gene activity for a complete gene. As such, they make it easier to diagnose various diseases that injmclude cancer.
Two main steps will be involved in the performance of the microarray lab experiments. These include the prehybridization and the hybridization steps. These are conducted through a number of 7 mini steps. They will involve the collection of the tissue or sample, the isolation of the RNA, isolation of the mRNA, creation of a labelled DNA copy, application off DNA, scanning of a microarray and the analysis of data (Campbell et al., 333).
Different pH indicators that are colorless at neutral and colored at high pH of above 10 will be applied. They will be mixed with molten Agarose; this includes Madison, Promega, WI and V312A. It will later be allowed to cool. They could also be placed in a hot bath of 650 and kept molten. They will be melted if to be used days later. Pipettes will be used to apply the DNA onto the slides.
Collection of mRNA
The plate will be incubated for 5 minutes to allow for the release of mRNA. It will then pipetted in a Tri reagent for extraction. 80 uL of chloroform will subsequently be added and shaken vigorously then centrifuged to separate the cells into layers. 2 ml isopropanol will be added, the mixture centrifuged and the supernatant poured off. After this, the preparation of the RNA for spec by will be done by adding Agarose gel.
The pre-hybridization steps will involve the preparation of stocks and obtaining of the microarray slide and steaming it on a hot plate for between 30 seconds and 1 minute. It will then be cooled at room temperature. It is important to warm the solution in case there are any crystals. The two slides can then be treated back to back and dipped in distilled water severally; it will be dried and spun for 2-3 minutes in a centrifuge. The slide is then hybridized by placing in a clean 50ml tube in a heated incubator. A coverslip is prepared by dipping into 0.2%SDS, then water. Blot, dry and continue to the hybridization step (Campbell et al., 338).
It includes the hybridization of the DNA chip using 3DNA array 350 protocol. Chips containing 70mer oligos and 2 copies of the known cDNAs in the human genome are used. This should be done at least 24 hours before the experiment. Make the solution only when it is ready for use. It is mainly 0.1M NaOH. The first step includes thawing vial at 7.2X. Make the hybridization solution with 50 ul total to fit across the cover slip. Incubate it at 800 for ten minutes. The entire 58 ul is then transferred on the microarray and the short edge of the cover slip placed on the short edge of the slide, which is then transferred to a 50 ml tube. The arrays after washing 2 must be read immediately since the color of the chips goes bad quickly (Kushner 1-5).
Campbell, A., Malcolm, Zanta, A., Carolyn, Heyer, J. Laurie, Kittinger,Ben, Gabric, M.Kathleen and Adler, Leslie. DNA Microarray Wet Lab Simulation Brings Genomics intothe High School Curriculum. CBE Life Science Education. 2006 Winter; 5(4): 332–339.
Kushner, B. David. DNA Microarrays in the Undergraduate Microbiology Lab: Experimentationand Handling Large Datasets in as Few as Six Weeks. Journal of microbiology andbiology education, 2007. Vol. 8