September 16, 2022
By: Alison Crandell and Jaxson Karline
After taking a break for the summer, we were eager to start our second year of RISEbio and continue working in the lab. Last year, we each picked out genes and hypothesized how their expression would vary across anole lizards’ breeding and nonbreeding seasons. After months of hard work, we successfully designed and tested primers for our genes and isolated RNA samples from the brains of anole lizards. To continue our work and ultimately examine the level of our gene’s expression in the brain, we must perform a quantitative PCR (qPCR). To do this, the first step is transforming the RNA we isolated last year into a more stable cDNA.
As we learned from previous semesters, RNA is highly unstable since it is more prone to hydrolysis and degradation than DNA. Therefore, we followed a two-step procedure to increase the likelihood of producing results during qPCR. The first step is turning the RNA we isolated into cDNA using Reverse Transcriptase PCR (RT-PCR), then using this cDNA for qPCR. For the first few weeks of class, we focused our skills on the first step of this process.
In RT-PCR, reverse transcription (RT) converts an RNA sample to cDNA. RT-PCR is different from a regular PCR since we are not targeting a specific sequence, and we are not amplifying the amount of DNA. We are merely converting the RNA present to cDNA and should have approximately the same amount of cDNA in the end as RNA that we started with.
Reverse transcriptase creates DNA using the RNA strand as a template, resulting in a complementary strand of DNA called cDNA. Since cDNA is synthesized from RNA, it only contains coding regions, as opposed to DNA which contains both coding and non-coding regions. Serving as a template for gene expression; therefore, cDNA synthesis is the first step for many protocols.
As this is the first step in a two-step process, we have not yet reached the step of producing documented results. But we are performing cDNA synthesis in the lab using a heat block at 65°C and an incubator heated at 42°C. A heat block has controlled heater technology, ideal for obtaining consistent results and precise temperature stability. An incubator is an insulated enclosure in which temperature, humidity, and other environmental conditions can be regulated at levels for optimal growth.
In a tube, we put nuclease-free water, RNA, and the primer for the RNA. We set the heat block at 65°C to allow our samples to denature. This removes any of the secondary structures of the RNA in our samples, such as RNA strands binding to themselves or other RNA strands. We then added the reaction and enzyme mix to the tube before placing it in the 42°C incubators for an hour, allowing the enzyme to attach and copy the RNA into cDNA. Afterward, we set the heat block to 80°C and placed our samples to denature the active transcriptase enzyme and stop the reaction. After completing the cDNA synthesis, the sample was put away in the freezer.
Throughout this process, everything went very smoothly, and we did not face any unforeseen issues. In the following weeks, we will continue to perform cDNA synthesis with more samples to accurately represent the male nonbreeding, female nonbreeding, male breeding, and female breeding groups of anoles in the qPCR. Additionally, we will help collect tissue samples from anole lizards that will be used for studies in the coming years. Many of these will be for the incoming RISEbio students who will pick their own genes to study just as we did, which seems so long ago after all that we have learned this past year.