RNA Isolations, ABHD2 and DPB Sequencing, and PALD1 PCR

12 April 2024

By: Paige Hoen, Hailey Ramthun, and Mady Docteur

Figure 1. Mady working on the PCR protocol for the PALD1 samples.

Over the past two weeks, we have continued to research these three genes within the Green Anole Lizards (Anolis carolinensis): PALD1, ABHD2, and DBP. Our goal is to discover the difference in gene expression between the breeding and non-breeding season. So far, we have had more success with our primers for ABHD2 and DPB. For each of these two genes, we identified a primer set that amplifies the correct band size on a gel. With the successful primer sets, we performed a PCR clean-up and sent our samples off for sequencing. Unfortunately, we have been facing difficulties finding a successful primer set for PALD1.

Figure 2. Gel image of RNA Isolation for a liver sample.

After numerous trials and errors with our first three primer sets for PALD1, we concluded it would be best to design new primers. When our new PALD1 primers arrived, we reconstituted them by adding water. Then, we performed PCR using each new primer set. After the PCR was completed, we loaded each sample into a gel and imaged it to see if the correct band sizes for each primer were amplified. Our results from the gel image showed primer dimers for two of the primer sets, and the other primer set showed hardly any bands. This means that we will continue troubleshooting with these primer sets. While working on PALD1 primers, we received our sequencing for DBP and ABHD2. The sequencing showed that our primer sets for both genes are good. Along with working on PALD1 primers, we have spent the past couple of weeks practicing RNA isolation. Eventually, a future step of our project is to perform a qPCR to analyze gene expression. We will need decent RNA isolation samples to complete this. The protocol for RNA isolation is slightly more complex than some of our previous protocols. This is because the RNA in the tissue can degrade if we do not work quickly enough. To practice with this protocol, we began by using liver samples in our first two attempts at RNA isolation. This was to make sure we knew what we were doing before using a brain tissue sample. After doing RNA isolation with liver tissue, we loaded our sample into a gel and imaged it. Both gel images showed that our RNA isolations were successful; showing two dark bands.

Figure 3. Paige and Hailey imaging the gel.

Since we successfully performed RNA isolation on liver tissue, our next step was to perform it again using brain tissue. Brain tissue thaws faster than liver tissue, so it was important that we worked quickly during the protocol. We completed our first brain sample RNA isolation. When we tested our sample on the Nanodrop, we got a concentration of 255.5 ng/┬ÁL along with a purity of 2.12. Purity should be close to 2.0, so we were pleased with these results. We ran a gel with the same sample. The gel showed two bands; therefore, we completed the RNA isolation successfully. We did another RNA isolation using brain tissue, and it had good concentration and purity. Next week, we will run a gel using this sample.

Our next steps for the remainder of this semester are to complete two more RNA isolations with a brain sample, ensuring we are consistently successful. Along with this, we will continue troubleshooting PCR for our three PALD1 primer sets. We will try PCR with a dilution, or a temperature gradient, to discover an effective PALD1 primer. An effective primer will amplify the correct band size in a gel electrophoresis with little to no primer dimers. Once we find a primer set that works, we will perform PCR cleanup on it and send it out for sequencing. Overall, we have made a lot of progress these last few weeks in the RISEbio lab. We have found two working primer sets, successfully performed RNA isolations, and are troubleshooting new PALD1 primers. These past weeks have not only taught us new lab skills, but the importance of staying on track, using time effectively in lab, and moving quickly to complete our tasks.

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