31 March 2023
By: Emma Rieper & Anna Wilcox
These past two weeks we have been testing our gene specific primers and performing RNA isolation techniques. We’ve had to conduct a lot of troubleshooting to get a proper result with our primers. There were also multiple new techniques and machines that we had to learn how to do/work with.
We started off last week, March 22, by performing an RNA isolation on a lizard liver sample. RNA isolation is a time sensitive, multistep process that begins with cleaning and using the homogenizer to grind up our liver sample and mix it with a QIAzol mixture. After homogenizing, we pipetted our sample into a microcentrifuge tube. We then added chloroform, followed by centrifuging, and pipetting off the aqueous layer on top. After adding 70 % ethanol to this, we used a RNeasy Mini Spin Column to allow the waste to spin to the bottom so we could discard it. After dumping waste multiple times, we had to centrifuge and dry the column. Throughout this experiment, we used two types of buffers, Buffer RPE and Buffer RW1. Water was then pipetted onto a column and centrifuged. Three microliters of sample were needed to perform quality checks. We also had to determine the RNA concentration and purity using the Nanodrop. After running a gel on this sample, we were able to see two clear bands.
To begin testing our primers we had to run a gel and see which primer worked the best. After choosing the best primer, we started a PCR clean-up protocol. First, we pipetted the rest of our sample into a new tube and added DF buffer (x5 volume). This was then centrifuged and the flow-through was discarded. Wash buffer was then added onto the column, centrifuged, and again the flow-through was discarded. In a new tube, Elution Buffer was added onto the column and centrifuged. We then had to measure the concentration and purity using the Nanodrop. We also had to make a small sample for Dr. Cohen to send off for sequencing.
Our RNA isolation overall went smoothly. However, there are a few tweaks we can make in the future to make the process smoother for when we start using lizard brain samples. As for our primer testing, we will be sending our sample off to DNA sequencing to find out if our primer is matching properly with the lizard genome. If our sample comes back inconclusive, we will begin working with one of our other primers, either primer A or primer B. From the last two weeks alone, we have learned the highs and the lows of research and how to regroup and create new plans for when experiments don’t turn out perfectly.