Results

Successful Results

• In vitro transcription of our crRNA and miRNA
• RNA purification of our crRNA and miRNA

Unsuccessful Results

• Initial functioning and proof of concept experiments

  • We performed variations of this experiment 5 separate times.
  • We believe that our lack of results was due to our inability to visualize our results. We attempted several versions of gel electrophoresis, altering gel percentage, amount of ladder/sample, amount of ethidium bromide, and introduced a denaturing step for the microRNA. We tried adding excess ethidium bromide into the TAE at the cathode end, and we attempted to let the gel soak in the TAE/EtBr mixture for readable results. Despite all of our efforts, none were successful.
  • Because our product size was so small (the miRNA was ~20bp, and smaller if the Cas13 became catalytically active), these gels were unable to display the small molecules.
  • If we were able to replicate this experiment again, we would have changed the type of gel we used instead of another EtBr agarose. Due to the size and funding of our lab, we were unable to obtain the necessary supplies to run a polyacrylamide, formaldehyde, or another gel that is able to more accurately and easily display miRNA.

• Gel #1

  

  Visible bands only in the DNA ladder lane. Our miRNA and experimental results are not visible.

• Gel #2

  

  Well #1 contained our Cas13+crRNA+miRNA reaction; well #2 contained just miRNA. The reaction mixture showed a bit of smearing, indicating possibly positive results. However, we cannot confirm that the smearing is due to successful cutting. We decided to run it again, this time with several other controls to help visualize and interpret the results. We also decided to add a denaturing step to prevent the miRNA from forming secondary structures and not separating properly in the gel.

• Gel #3

  

  8 of the 10 wells contained a product, with the first well being an RNA ladder. The ladder showed unsuccessful separating, indicating an issue with our gel or with excess ladder used. We also noticed the EtBr began running off the gel, cutting off visibility where we would see our results. We ran it again to correct the EtBr and ladder issues.

• Gel #4

  

  Ladder separated successfully. No product visible in any other well. This gel also contained random splotches of illumination. We did not denature the product for this gel, so we ran the experiment one last time to include that step.

• Gel #5

  

  Only visible bands in the ladder lane. We suspect that the gel ran for too long, causing our small product to run off the gel. If we had the time, we would run this again for a shorter amount of time.

Future Directions

Due to the timing of our project, we were unable to complete any more experiments. However, we have several directions we want to go with our project.

• Our fourth experiment after concluding that the miRNA is degraded by Cas13, would have tested the specificity of the enzyme by also adding other miRNA sequences and comparing the results. This would ensure that the Cas13 will only activate in the presence of miR-424-5p and no other miRNA, lowering the probability of false positives. We attempted this experiment on our last gel, however the gel showed no meaningful results.
• At this point, we planned to introduce our quencher fluorophore signal. When added to the experiment, the bond holding the two molecules together would be cleaved by catalytically active Cas13, causing the solution to fluoresce. This is the signal that would alert the consumer if miR-424-5p is detected in the urine.
• We discussed running this system with synthetic urine to test if other molecules in the urine would affect how the experiment runs.
• Lastly, once our system was proven to work, we aimed to test how our device would hold up when in a consumer’s possession. Several factors can impact the stability of our system, so we planned to test the effects of a few conditions: the number of freeze-thaw cycles, the storage temperature, the buffer solution used, and light vs. dark storage.