April
17th, 2019
By Kaela Goodman, Meaghan Keohane, and Ramsey Pankratz
Introduction
The goal of our research is to predict whether or not certain miRNAs regulate the expression of the APOBEC3A and APOBEC3B genes. Once the regulatory relationship has been demonstrated, a future step would be to identify if that miRNA is present inside tumor cells at unusual concentrations, and incorrectly regulating the expression of either gene.
So far, our research has been focused on developing usable miRNA samples. To start, we researched and picked out three microRNA sequences that were predicted to bind to either APOBEC3A or APOBEC3B. For our group, the selected miRNA are hsa-miRNA-4786-3p, hsa-miRNA-4522, and hsa-miRNA-890. The following steps include finding usable forward and reverse primers, amplifying each of our miRNA using polymerase chain reactions (PCR), and then purifying our PCR products. So far, we have developed usable concentrations of amplified hsa-miRNA-4522.
Figure 1 The chromosomal location of hsa-miR-4522 in the human genome at 17q11.1. From http://ghr.nlm.nih.gov/chromosome/17
Our Process
This week, beginning with our purified PCR product, we successfully performed ligation and transformation. For the first part of this process, ligation, we inserted hsa-miRNA-4522’s PCR amplicon into the vector, pMiniT 2.0, to create a plasmid. To achieve this step, all we need to do was carefully mix the vector, our purified PCR product, and two cloning mixes together. Then simply allow them to incubate to create multiple identical “children” plasmids.
Next, we attempted to transform the plasmids into E. coli cells. We added our ligation reaction to a tube containing ampicillin resistant E. coli and then attempted to trick the E. coli cells into taking up our plasmid by heat-shocking them. The heat-shock created an environment that made the E. coli think that it was about to die so it would take up anything around it, like our plasmid, to prevent its death. We then plated two samples of the heat-shocked cells, one regular and one tenth dilution, on plates of lysogeny broth media with ampicillin (LB-Amp plates). Finally, we allowed them to grow, in an incubator, overnight.
Figure 2 Meaghan carefully plating one of the E. coli samples.
The success of the ligation and transformation was determined by the growth, the number of colonies on the plates. It was determined that if the created plasmid was able to successfully be transformed, the E. coli cells would grow on the LB-Amp plates. Why would the growth of colonies indicate successful transformation? It is due to the fact that the vector contained a lethal minigene that, without our insert, would kill the cells. However, if anything was able to be inserted into the vectors, theoretically, our amplicon, it prevents the translation of the lethal minigene, which would allow growth of the cells. When we checked our plates the next morning, both plates grew well. As expected, our undiluted plate had many more colonies in comparison to the one-tenth dilution.
Figure 3 Ramsey counting the colonies on the one-tenth dilution plate.
Future Steps
Working towards developing an understanding the role of APOBEC3A and APOBEC3B genes in the development of cancer, after growing our plates, we will do a number of steps to move forward in our research. After plate growth has been identified, we will isolate 6 separate colonies from our plates, and purify them. Then, a restriction digest will be performed to separate the inserted fragment (our miRNA sample) from the pMiniT 2.0 vector. After interpreting our results for the restriction digest, we will determine if the process was successful, the sample will be sent off for sequencing. Sequencing will allow us to successfully identify if the sample we have been working with is actually the miRNA we think it is.
