Mentor: Dr. Stark

Location of Research: DNA Learning Center at Notre Dame

Category of Research: Molecular Biology

Working Title: Differential Induced Gene Expression in Platinum Based Chemotherapy

Topic/problem: ā€¨Comparing the gene expression of 4 gene after cisplatin or carboplatin treatment in hopes to gain a better understanding of why some patients respond to one of these drugs, but not the other, though cisplatin and carboplatin are very similar drugs.

Research Plan: A549 lung cancer cells will be grown in cell culture. The cells will be treated with the IC50 concentration dose for cisplatin or carboplatin at 0, 3, and 18 hours. The drug treatment will be done in triplicate. The cells will be harvested at the time points and the mRNA will be extracted and reverse transcribed to cDNA. The gene expression of CASP3, ERCC1, and XAB2 will be measured using quantitative Real-Time PCR (and normalized to a housekeeping gene). qRT-PCR will be performed twice in triplicate. Statistical analysis will determine differential expression at each time point.


Comments:

Meetings:

8/27/15
Met with Dr. Stark. We discussed possible project topics for this year.

8/31/15
Met with Dr. Stark. We continued to discuss possible project topics for this year.

9/16/15
Met with Dr. Stark. We continued to discuss possible project topics for this year. We looked at the calendar to get a better view of the time frame for this project.

9/30/15
Met with Dr. Stark. Began finalizing my new project and brainstormed for a hypothesis by reading articles on cisplatin and carboplatin.

10/2/15
Met with Dr. Stark. I made a general outline of my project and discussed the grant application with Dr. Stark.

10/13/15
Met with Dr. Stark. I went over some questions about the grant application.

10/14/15
Met with Dr. Stark. I finalizd the grant application and we filled out ISEF paperwork.

10/21/15
Met with Dr. Stark. I practiced cell culture techniques on breast cancer cells that were currently in the lab. I removed current media and added 1mL of trypsin. I put the cells in the incubator for 5 minutes to activate the trypsin. Once cells were detached, I added 2mL of media to deactivate trypsin and moved 2mL of the cells to new flask. I added 6mL of new media to the flasks and put them back in the incubator.

10/30/15
Met with Dr. Stark. I worked with my A549 lung cancer cells in cell culture. I removed the media from the only flask that I had. I washed the flask with PBS and added 1mL of trypsin. I incubated the cells for 5 minutes and added 2mL of media to deactivate the trypsin. I removed 2mL of cells and divided them into 2 flasks. I added 6.5mL of fresh media to all 3 flasks and put them in the incubator.

11/4/15
Met with Dr. Stark. I removed media from all 3 flasks. I added 1mL of trypsin to detach the cells and incubated the flasks for 5 minutes. I added 2mL of media to each flask to deactivate trypsin. I removed 2mL of cells from each flask and put them into 2 new flasks with 1mL of cells in each to get a total of 9 flasks. I added 6.5mL of media to each flask and put them into the incubator.

11/5/15
Met with Dr. Stark. I prepared flasks for drug treatment to be done on 11/6/15. I removed media from all 9 flasks. I added 1mL of trypsin to detach the cells and incubated the flasks for 5 minutes. I added 2mL of media to deactivate the trypsin. I removed all 3mL of cells to empty the flasks and put 1mL of cells into 3 new flasks to get a total of 27 flasks. I added 6.5mL of media and incubated the flasks.

11/6/15
Met with Dr. Stark. I weighed out the drugs cisplatin and carboplatin 3 separate times in order to do the drug treatment in triplicate. I calculated the amount of DMSO (vehicle) needed for the amount of each drug and made the treatment. I took 7 flasks out of the incubator. I removed all of the media from the flasks except 5mL. I treated 2 of the flasks with 5mL of cisplatin treatment, 2 flasks with 5mL of carboplatin treatment, 2 of the flasks with 5mL of the vehicle treatment (DMSO and media), and 1 flask with just 5mL of media. I set up 7 flasks like this 3 times (drug treatment in triplicate). For the 0 hour time point, I took the 3 flasks with just 5mL of media. I removed the media and added 1 mL of trypsin to detach the cells. I incubated the flaks with trypsin for 10 minutes or until the cells detached. I added 2mL of media to deactive the trypsin. I washed the flasks and moved all of the cells from the flasks into seperate Falcon tubes. I centrifuged the 3 tubes to form a cell pellet and removed all of the liquid. I froze the cell pellets in a -80 degree Celcius freezer. For the 3 hour time point, I took 1 flask treated with cisplatin, 1 flask treated with carboplatin, and 1 flask treated with vehicle from the 3 drug treatments that I set up giving me a total of 9 flasks for the 3 hour timepoint. I removed the media from each flask, added 1mL of trypsin to detach the cells, incubated the flasks for 10 minutes, added 2mL of media to deactivate the trypsin, washed the flasks, removed the cells from each flask and put them into a Falcon tube, centrifuged the 9 tubes, removed the liquid from the cell pellet, and stored the cell pellets in a -80 degree Celcius freezer. The 18 hour time point cells will be harvested tomorrow.

11/7/15
Met with Dr. Stark. I harvested the cells for the 18 hour time point treated with cisplatin, carboplatin, and the vehicle. I removed the media from each flask,
added 1mL of trypsin to detach the cells, incubated the flasks for 10 minutes, added 2mL of media to deactivate the trypsin, washed the flasks, removed the cells from each flask and put them into a Falcon tube, centrifuged the 9 tubes, removed the liquid from the cell pellet, and stored the cell pellets in a -80 degree Celcius freezer.

11/30/15
Met with Dr. Stark. We went over the methods for the next step of the project which is isolating that mRNA and reverse transcribing it to cDNA. We will begin the process on Wednesday.

12/9/15
Met with Dr. Stark. I isolated the mRNA from the cells following the protocol from the mRNA isolation kit. I nanodropped my samples to make sure I had collected enough mRNA.

12/11/15
Met with Dr. Stark. I reverse-transcribed the mRNA to cDNA following standard protocol.

12/14/15
Met with Dr. Stark. I aliquoted cDNA and created cDNA standard curve pool.

12/16/15
Met with Dr. Stark. I mapped out the plates that I will be running in the PCR machine.

1/11/16
Met with Dr. Stark. I prepared a 96 Well Plate with cDNA dilutions and the primer for the housekeeping gene B2M. I ran it through the PCR machine.

1/13/16
Met with Dr. Stark. I prepared a 96 Well Plate with cDNA dilutions and the primer for the housekeeping gene B2M again. I ran it through the PCR machine.

1/14/16
Met with Dr. Stark. I prepared a 96 Well Plate with cDNA dilutions and the primer for the gene XPA. I ran it through the PCR machine.

1/15/16
Met with Dr. Stark. I prepared a 96 Well Plate with cDNA dilutions and the primer for the gene CASP3. I ran it through the PCR machine.

1/18/16
Met with Dr. Stark. I prepared a 96 Well Plate with cDNA dilutions and the primer for the gene ERCC1. I ran it through the PCR machine.

1/19/16
Met with Dr. Stark. I ran a 96 Well Plate with cDNA dilutions and the primer for the gene ERCC1 again. I also ran another 96 Well Plate with various primers to gain more confidence in some of my results.

1/20/16
Met with Dr. Stark. I exported my data for all of the plates I ran and analyzed the information. I made rough sketches of the gene expression graphs.

1/25/16
Met with Dr. Stark. I ran a 96 well plate with cDNA diluations and the primer for the gene PARP. I ran the plate in the PCR machine.

1/27/16
Met with Dr. Stark. I analyzed data from the 96 well plate with the PARP primer and made a rough sketch of the gene expression graph.

2/4/16
Met with Dr. Stark. We discussed the tri-fold and how to set it up.

2/8/16
Met with Dr. Stark. I practiced presenting my research.

2/15/16
Met with Dr. Stark. I practiced presenting my tri-fold.

2/22/16
Met with Dr. Stark. I finalized my regional poster board.

2/23/16
Met with Dr. Stark. I went over last minute items on my poster board.