Experiments

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.

Protocol:

(page 6)

Set-Up

• Added 1125 µL of MilliQ water to 2023MissouriMinersT7CRRNA424 (crRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
• Added 615 µL of MilliQ water to 2023MissouriMinersT7MIRNA424 (miRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
• Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
• Keep TranscriptAid Enzyme Mix and nucleotides on ice.
• Keep the 5X TranscriptAid Reaction Buffer at room temperature.
• Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

1. Combine the following reaction components at room temperature in the order given:

   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)

2. 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.

Protocol:

1. Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiments up to 100 µL.
2. In two 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
3. Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
4. A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load the lysate into the RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
5. For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
6. For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard the flowthrough and place the column back into the Collection Tube.
7. For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease-free Collection Tube
8. Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
9. Store the RNA at -20°C until ready to use

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.

Protocol for Reaction tubes:

1. 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.

2. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
3. Incubate the solution at 37 C for 1 hour in a water bath.
4. Remove the tube from the water bath and add 9 uL of miRNA to the solution as well as enough Milli-Q to bring it to 20 uL and mix carefully by gently inverting the tube.
5. Incubate the solution again at 37 C for 10 minutes.

Protocol for Current Gel Electrophoresis Method:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. Carefully and slowly pull the comb straight up and out of the gel.

12. 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

13. Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
14. Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
    1. Incubate in a 70°C water bath for 10 minutes.
    2. Chill samples on ice for 3 minutes and spin briefly.
15. Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
16. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
17. Put the gel on a UV illuminator to see the results.