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 spatula should be sterile. Once the spatula is sterile, it is to be used to cut a 1-cm square of agar with worms from the wild type starter plate and transfer the chunk to the OP50 seeded plate that is labeled “wild type”, face down. This same procedure is repeated to transfer worms from the dpy-13 starter plate to the OP50 seeded plate labeled “dpy-13”. These plates are then both incubated upside down at 20˚C for 48 hours. This incubation gives the worms time to grow in their new environment.
In part 2 of the experiment we are inducing RNAi through the process of feeding. To do so, a black pen is used to label the bottom of an OP50 seeded plate with the date and “wild type”. Another OP50 seeded plate is labeled with the date and “dpy-13”. A black pen is then used to label the bottom of the plate seeded with dpy-13 RNAi feeding strain with the date and “wild type”. Five L4-stage worms from the plate of wild type worms are then picked and moved to the OP50 seeded plate labeled “wild type”. Any eggs or young larvae that may have been accidentally transferred are to be picked off of the plate and then flamed in a Bunsen burner. The same method is used to move five L4 wild type worms to the plate seeded with the dpy-13 RNAi feeding strain, and once again to move five L4 dpy-13 worms to the OP50 seeded plate labeled “dpy-13”. These plates are then incubated upside down at 20˚C.
Part 3 of this experiment calls to isolate the DNA from C. Elegans. To do so, we start by labeling three PCR tubes with the group number. One of the tubes is labeled “W” for wild type, one is labeled “R” for RNAi, and one is labeled “D” for deletion mutant. Once they are labeled a micropipette, with a fresh tip each time, is used to add 10µL of lysis buffer to each of the tubes. Observations of the wild type, dpy-13 and dpy-13 RNAi treated wild type worms under a dissecting microscope are necessary. Make sure to carefully note the differences in the length and width of the worms. Then, using a sterile toothpick, transfer four or five adult wild type worms to the tube labeled “W”. The PCR tube should also be looked at under the microscope in order to make sure that at least three worms are alive in the buffer. Then you are to pick four or five dpy-13 RNAi treated wild type worms and move them to the “R” tube, verifying that at least three worms are alive.
Then you are to pick four or five dpy-13 worms to the “D” tube, verifying that at least three live worms exist. The PCR tubes are then fit into one or two adaptors. The PCR tubes are “nested” into larger adaptors: a 0.2 mL tube, within a 0.5 mL tube within a 1.5 mL tube. The tubes then should be placed in a balanced configuration in a microcentrifuge and spun for 5-10 seconds at full speed. This process will pellet the worms. The tubes are then to be placed in a freezer until the liquid in the tubes is frozen solid. The freeze/thawing cracks the tough outer cuticle of the worm. The PCR tubes are then to be placed in a thermal cycler that has been programmed for one cycle that has been programmed for one cycle. The cycle should contain an incubation step that is at 65˚C for 90 minutes followed by a boiling step at 95˚C for 15 minutes. The sample should be stored on ice or at -20˚C until the next step of the experiment is ready to be performed.
In part 4 of the experiment you are to amplify the DNA through PCR. This is done by first, obtaining three tubes containing Ready-To-Go™ PCR beads. Each tube should be labeled with the group number. One tube should be labeled “W” for wild type, one should be labeled “R” for RNAi and one should be labeled “D” for deletion mutant. A micropipette is then used, with a fresh tip each time, to add 2.5µL of worm DNA from part 3 to the appropriate tubes.
The DNA should be added directly into primer/loading dye mix, no worm DNA remains should be in the tip after pipetting and a fresh tip should be used for each reaction. The next step is the thermal cycler, which should contain the cycles or a denaturing step at 94˚C for 30 seconds, an annealing step at 55˚C for 60 seconds, and an extension step at 72˚C for 60 seconds. After the cycling the amplified DNA should be stored on ice or at -20˚C until part 4 of the experiment is ready to be begun.
Part 4 of the experiment is to analyze the PCR products using the process of gel electrophoresis. A gel is provided in the lab to be used. Using a micropipette add, with a fresh tip each time, 20µL of pBR322/BstNI size marker into the far left lane of the gel. Using a micropipette, with a fresh tip each times, add 20µL of each sample/loading dye mixture into a different lane of the gel, the marker being in well “1”, wild type should be put in well “2”, RNAi treated dpy-13 in “3” and Deletion dpy-13 mutant in “4”. The gel is to be run at 130V for approximately 30 minutes. The gel is then to be stained in ethidium bromide.
Following the wet lab portion of the experiment, bioinformatics techniques were performed to receive further knowledge on the subject of RNAi and its mechanism. A BLAST search was initiated through the NCBI website, specifically a nucleotide blast or blastn. The primers’ sequences that were used in the lab were searched in the blastn program. The blastn sequence was first optimized for similar sequences and then the sequence was then analyzed using the program. Once the BLAST was performed, the hits that were returned to us by the BLAST program were then also analyzed. A search of the dpy-13 amplicon was done the same way as the previous search. However, this time, a megablast is performed. The dpy-13 amplicon was also analyzed.
Observations of the Wild Type and dpy-13 mutant strain of the C. Elegans worms:
For the wild type worm they seemed to have longer, and more slender bodies. Also, they had much faster movement on the plate. They seemed to have an easier time moving around on the plate. For the dpy-13 mutant plate they seemed to have a shorter, fatter body. Also, their movement was much slower. Not nearly as fast moving as the wild type worm. When the worms were treated with the RNAi they also had shorter, fatter bodies and were not as active and slower moving. The table below shows pictures of each of the types of worms taken through a microscope and cropped at the same size: Wild Typedpy-13dpy-13 RNAi treated
The sample below of the gel electrophoresis was taken from the book:
The first well on the left being the marker, next the wild type, then the RNA treated dpy-13, then a dpy-13 deletion mutant, then ladder
The sample below of gel electrophoresis is the gel electrophoresis that was performed from the samples from the experiment:
The marker being is the first well on the left, next being the wild type worm, and then the RNAi treated wild type, and last the dpy-13 deletion mutant.
Discussion and Conclusion:
After observing the dpy-13, wild type, and the RNAi treated C. Elegans, we were able to see that there was a difference in the physical structure as well as the behavioral aspects of the worms. Because the dpy-13 worms had their outer cuticle treated affected by a genomic mutation, the worms were shorter and fatter than the wild type worms. Also, the dpy-13 worms moved much slower than the wild type worms. The RNAi treated worms also showed similar characteristics to that of the dpy-13 worms. We were able to determine the physical and behavioral characteristics by simply observing the worms through a microscope, however, to determine the genotypic differences we were required to isolate the DNA from each sample.
We were able to isolate the DNA by performing a PCR reaction. From the PCR reaction we were able to perform a gel electrophoresis. Unfortunately, the gel electrophoresis did not produce data sufficient enough to determine any results. The reasons for this could range from damage during transfer, an error during the PCR reaction, or even an error that could have occurred during the storing of the worms. Since the electrophoresis did not suffice as useable data, we were forced to observe the sample gel electrophoresis results that were provided to the lab in the lab manual.
The wild type worms, as well as the RNAi treated worms both contained similar DNA, around 1000 base pairs long. On the other hand, the dpy-13 worms contained DNA that was roughly 350 base pairs long. From these observations of the gel electrophoresis results we were able to conclude that even though the wild type and the RNAi treated worms contained the same DNA composition, because the RNAi mechanism resulted in a difference in the phenotype of the worm that resembled the dpy-13 worms, the results support that the RNAi treated worms went through a type of post-transcriptional modification, specifically, post-transcriptional gene silencing that most likely silenced the same gene that the dpy-13 mutant deleted.
There are possibilities of future experiments that can be conducted. Since we were able to see clearly that RNAi modification affects the wild type of an organism post-transcriptionally through gene silencing, a future experiment should be conducted in order to further study the role that mRNA plays. Reverse transcriptase is used to produce RNA from DNA. To isolate the RNA that is used, a PCR reaction can be done in order to isolate the specific mRNA. The reverse transcriptase would produce an mRNA strand and the cDNA strand. If RNase is added to the solution, the mRNA can be broken down. From there, a DNA polymerase, dNTP’s and a primer are used to produce new copy strands of the cDNA already present and then the total RNA can be isolated.
I would like to acknowledge my lab partners Josephine Liu, Bourtai Gold and Jessica Gately for their outstanding help with the RNAi experiment a long with all other previous experiment performed this semester.
“Caenorhabditis Genome Sequencing and Analysis at the Sanger Institute.” Caenorhabditis Genomics. N.p., n.d. Web. 05 Dec. 2012.
“Introduction to C. Elegans.” Introduction to C. Elegans. N.p., n.d. Web. 05 Dec. 2012.
Lewis, Susan K. “The RNAi Cure.” PBS. PBS, 01 July 2005. Web. 05 Dec. 2012. Pierce, Benjamin A. Genetics: A Conceptual Approach. New York: W.H. Freeman, 2012. Print. “Silencing Genomes.” Silencing Genomes. DNA Learning Center, n.d. Web. 03 Dec. 2012. .