{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %} {% block page_content %}
We utilized public databases such as NCBI, the National Institute of Health, and the National Library of Medicine to search for papers and design protocols; identified biomarkers by utilizing these papers to find RNA biomarkers commonly found in breast and other cancers; and searched for our RNA sequences using RNAcentral. We selected several other variants of miRNA such as miR-320c, miR-10b-5p, miR-21, and miR-16 to test because these miRNAs are either prominent miRNAs found in urine or have been identified as potentially useful for breast cancer diagnosis. To confirm the results of our experiment we decided to use electrophoresis to detect miRNA bands within our sample. This will test whether or not they had been cut by our Cas13 enzyme or not.
Our first protocol was in-vitro transcription to produce the miRNA and crRNA we would use for our experiments. We used a T7 TranscriptAid kit for the in vitro transcription.
(page 6)
Compoent | Amount |
---|---|
DEPC-treated water | to 20 µL → 5.5 µL |
5X TranscriptAid Reaction Buffer | 4 µL |
ATP/CTP/GTP/UTP mix* | 8 µLb |
Template DNA | 2 µg → 0.5 µL |
TranscriptAid Enzyme Mix | 2 µL |
Total volume: | 20 µL |
* Equal volumes of the four provided NTP solutions combined in one tube.
Tubes: (miRNA, trans, 4/20) and (crRNA, trans, 4/20)
Mix thoroughly, spin briefly to collect all drops, and incubate at 37°C for 4-8 hours (short transcript).
Our second experiment’s purpose was to purify the transcription products from our first experiment. For this experiment, we utilized Nucleospin and a centrifuge to separate the transcription products from the rest of the solution. The tubes appeared more transparent than initially, indicating some level of purification.
We measured the concentrations of RNA on a nanodrop and found that the final concentration of crRNA was 25.6 ng/ul, and the final concentration of miRNA was 8.8 ng/ul.
The third experiment tested if the target sequence was cut. For the protocol, we utilized electrophoresis to attempt to detect if our Cas13a enzyme had cut the miRNA. We were unable to visualize the miRNA in our gel. From these results, we realized we needed a more sophisticated gel and a higher concentration of RNA to better see the bands. Further testing revolved around creating a gel electrophoresis protocol that would allow us to visualize the bands of very small ( <20 nt ) degraded miRNA.
Component | Starting Concentration | Amount |
---|---|---|
Milli-Q Water | 6.5 ul | |
crRNA | 25.6 ng/ul | 0.5 ul |
Cas13 Enzyme | 63.3 ug/ul | 2 ul |
Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | 10 X | 2 ul |
miRNA | 28.4 ng/ul | 9 ul |
Total Volume: | 20 µL |
Label 7 tubes as shown below:
miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 |
---|
All tubes will have Milli-Q water and reaction buffer. The labels above indicate what will be added to each tube for the reaction.
Prepare a chart to keep track of which sample you will place in each well.
RNA Ladder | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
---|