Assessing Differential Gene Expression of Drosophila Melanogaster Using Differential Display Technique

This research project was intended to analyze the differences in gene expression at different life stages of drosophila melanogaster by using a differential display approach. I focused on the adult and pupae stages. The gene expression of the larvae and egg stages were not analyzed because the larvae and eggs were too small to collect. First I isolated RNA from two different generations of adult flies (separately), young adults (recently eclosed adults), and pupae. Then I synthesized cDNA by reverse transcription in the PCR. The cDNA was then amplified in a PCR reaction. Next, I ran the amplified cDNA in a denaturing polyacrylamide gel. The gel did not come out as well as expected: few bands were visible. The bands that were visible, those of the pupae sample, came out as expected. The bands of both stocks of adults and the young adults did not come out. This is probably because not enough cDNA was present. Next term I plan to isolate RNA from more flies (maybe six instead of two) in order for a larger quantity of RNA to be extracted. Then more cDNA would be synthesized and amplified. I will also change the MgCl2 and dNTP concentrations of the PCR amplification reaction in order to achieve optimal results. If bands are shown, I will cut the bands of interest out of the gel, reamplify them and sequence them. Taking in consideration the technical difficulties of the procedures involved in this project, I think overall it was successful.