February 25, 2020
By: Taylor Forstner & Emilie Greene
Introduction:
Our overall goal in the Immunity and Cancer Research Stream is to determine the relationship APOBEC3A and APOBEC3B have with the progression of cancer, and how miRNAs effect and inhibit the expression of these genes. In past weeks, we have successfully run protocols giving us reliable controls. Now we begin to research and test specific miRNAs that could bind and regulate these APOBEC genes.
Research:
Using the extracted HeLa DNA and the successful PCR amplification of hsa-miR-425 done in past weeks, we could begin to research and determine what specific miRNAs we wanted to continue with. Using an online miRNA target prediction database, we chose two miRNAs based on their targeted score which reflects whether or not they would bind successfully to APOBEC3A, APOBEC3B or both. We chose hsa-miR-4303 and hsa-miR-4297. Hsa-miR4303 binds only to A3A (target score 88), while hsa-miR4297 binds to both A3A and A3B (target scores 83 and 82 respectively).
Taking these miRNAs, we used GenBank at NCBI to obtain their exact sequences. After modifying the sequences to get the ideal length, we used Primer3Plus to design and order primers that should bind and amplify our chosen miRNAs.
Primers are small pieces of single stranded DNA that are used to flank the targeted miRNA that we want to amplify. They control what segments get duplicated and thus what we visualize during gel electrophoresis.
The PCR process began with creating Master Mixes that contained all the components needed for successful amplification. These Master Mixes contained, PCR water, Q5 High-Fidelity 2x Master Mix, and the forward and reverse primers corresponding to the miRNAs we wanted to amplify. In total we had three miRNA to test; the hsa-miR-425 obtained in previous weeks as a control, and the two miRNAs researched: hsa-miR-4303 and hsa-miR-4297. We ended up with six samples in total: three that contained DNA template and should show bands in the final gel, and three no DNA controls that should show no bands.
Part of PCR is running samples through a thermal cycler which subjects the samples to varying cycles of different temperatures. These different temperatures all serve a unique purpose, some denaturing the DNA and others promoting the amplification. One crucial temperature is the annealing temperature that allows the primers to successfully bind to the DNA, which can later be duplicated. Each primer has a different annealing temperature, so using New England Biolabs’ TM Calculator, we were able to find the correct annealing temperatures for our two unknown primer pairs.
The PCR was placed through the thermal cycler, and the amplified samples run through gel electrophoresis. Our first gel was inconclusive, so another one was run using the same samples. This gel was imaged and then analyzed, looking for strong bands in our DNA samples, and no bands in the controls.
Reflection:
Our primer design, PCR, and gel electrophoresis all concluded with positive successful results. We observed that the control hsa-miR-425 PCR was successful, and our two primers we designed resulted in the amplification of our miRNAs. Our only setback was minor; with our first gel not giving conclusive results, but that was easily solvable by running another gel. Our next steps will be to purify the PCR products obtained in this protocol.
