November 9th, 2021
D’Aaliyah Johnson, Matthew Trenne, Carter Beck
Introduction:
Last Semester, in the spring of 2021 we chose two miRNAs to work with and investigate their effect on APOBEC3A and APOBEC3B, these miRNAs were hsa-mir-4297 and hsa-mir-1207-3p. In our last post we had just finished a restriction digest to cut our microRNAs out of the pMiniT that we had placed them in earlier. After that step, all three of us were extremely excited to continue to the finish line that was the dual luciferase assay
Specific Content:
Our second restriction digest did not show the results that we were looking for, so we unfortunately had to take a step back and reevaluate what was going on and potential problems. Looking forward we decided to add more of our miRNAs to the mixture being run in the restriction digest by doing a plasmid rescue protocol. We had to perform another plasmid and bacterial transformation so that we had more plasmid to work with. We did this, ran another restriction digest, and saw exactly what we wanted to see as far as concentration of our miRNAs and the amount of miRNA present in each sample.
Next, we took the gel that was made and imaged after the restriction digest and used the UV (ultraviolet) light box to cut out the bands what were present at 300 bp for the 1207-3p and 4297 samples. This involved lighting up the UV box for short periods of time to see the bands then cutting around the bands to remove them from the gel.
After that, we worked on a ligation and transformation protocol. The goal of this step was to ligate the miRNAs into the pcDNA and transfer them into bacteria. We started with the ligation protocol and after that we plated the product on three different plates for each sample at three different concentrations as well as control plates. We saw only one colony grow on the control plates, but other than that there was great growth on the plates with the active miRNA.
Our next step was to inoculate our colonies and prepare them for mini prep and subsequent sequencing. We needed to inoculate our colonies to that we had specific samples to be able to send out, we ended up sampling four colonies from both 1207-3P and 4297. For the mini prep and sequencing we nano dropped the samples to determine concentration of pcDNA and our data was amazing, seeing concentration numbers well into the two hundreds and above when the goal was to get them in the hundreds.
Reflection:
Our goal is to successfully run a luciferase assay and have that data show whether miRNA 1207 and miRNA 4297-3 are active in regulating APOBEC3A and ABOPBEC3B, respectively. We will be running this assay two times to both give us more data to work with and to work as a control against each other. This luciferase assay is the final step in our research stream, we have worked extremely hard and put in countless hours in the lab to get to where we are today.
