Examining the Rna Interference Mechanism in the Dpy-13 Gene in C. Elegans Through Feeding

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Examining the RNA Interference Mechanism in the dpy-13 Gene in C. Elegans Through Feeding Mehdi Misto
Lab: Monday 1:00 – 4:50 PM
11 December 2012

Introduction:
RNA interference, or RNAi, is a biological process in which RNA molecules reduce the gene expression of an organism. This is done typically by causing the destruction of specific mRNA molecules. RNAs are direct products of genes, these small RNAs can bind to other mRNA molecules to either increase or decrease their activity like in the example of preventing an mRNA from producing a protein. There are two types of RNA molecules that are central to RNAi, these molecules are, micro RNA (miRNA) and small interfering RNA (siRNA). The RNAi mechanism is found in many different eukaryotes and it is started by an enzyme names Dicer. Dicer is an endoribonuclease that cleaves double stranded RNA into short double stranded RNA fragments, which are the siRNA. Each of these new siRNAs are then unwound into two different single stranded ssRNAs. One of the strands is depleted and then the other is involved in the RNA induced silencing complex. RNAi is a very valuable in research. This is because double stranded RNAs that are introduced into the cell can induce suppression of some specific genes of interest. Andrew Fire and Craig C. Mello’s work on RNAi in C. Elegans revolutionized RNAi, and because of it the usefulness of it has increased substantially. RNAi can be easily introduced into the C. Elegans through feeding; feeding the worms the bacteria that expresses double stranded RNA that corresponds to the gene that is being targeted can do this.

In this experiment, to introduce the C. Elegans to the RNAi, the worms were first transferred to a plate containing the IPTG. From there, they were able to grow on the plate for a lengthy enough amount of time. Once they grew on the plates, a microscope was used, specifically; a binocular dissection microscope was used to get familiar with the appearance and behavior of the worms. Determination of whether or not the RNAi was successfully induced in the worms needs to be done next. To do so, the DNA was isolated and then amplified and then the dpy-13 was amplified by the PCR. Once the DNA was amplified, it was analyzed by gel electrophoresis. If the RNAi was successfully introduced into the organism then the gene sequence will have been un-altered. The only thing that should differ in the C. Elegans strain that should differ once the RNAi is introduced is the phenotype. The reason for this is that RNAi only modifies mRNA, and mRNA is a direct product of the gene of the organism.

Materials and Methods:
To begin this experiment, first a proper introduction to the material is necessary. This is started with a few online websites that are selected for the lab partners to visit and research. Research about RNAi is done on the website www.pbs.org/wgbh/nova/body/rnai-cure.html as well as a little research done on Wikipedia. Research about C. Elegans is done on the websites http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html, and http://www.sanger.ac.uk/research/projects/caenorhabditisgenomics/. The website http://www.silencinggenomes.org/ is also used to research the mechanisms and background of RNAi. In order to make sure that research is done properly, a worksheet is handed out to the class by the professor to direct the lab through the website and ask certain questions to promote the learning that is necessary to continue on with the lab.

In part 1 of the experiment we are transferring wild type and dpy-13 C. Elegans to OP50 seeded NGM0lite plates. To do so, a Wild-Type C. Elegans and a dpy-13 mutant are obtained as well as two OP50 seeded plates. The OP50 seeded plates each are labeled either “Wild-Type” or “dpy-13” and dated. In order to sterilize a metal spatula, it is dipped into ethanol and then briefly passed through a Bunsen flame to ignite the alcohol. Once the alcohol is burned off the metal...
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