Materials:

• Direct-zolTM RNA Miniprep Kit from Zymo Research
• TRI Reagent (or Trizol)
• Liquid nitrogen
• Absolute Ethanol (200 proof)
• Micropipettes (p10, p100, p1000)
• 1.5 mL RNase/DNase-free tubes
• 1.5 mL tube pestles
• Fixed-angle centrifuge
• NanoDrop Spectrometer
• Qubit4 Fluorometer
• Qubit Assay Tubes
• Qubit brRNA standards and reagents (kit)
• Glass petri dish
• 70% ethanol (regular ethanol)
• B. glabrata (NMRI strain) snails (1 cm shell size)
• Tweezers (possibly a dissection kit)
• 250 mL liquid waste beaker
• 100 mL beaker
• Kimwipes
• Vortexer
• Sharpie marker

Protocol:

RNA Extraction from B. glabrata (NMRI) snails:

1. Prepare all reagents in Direct-zol RNA Miniprep Kit prior to beginning extraction.
2. Obtain 1 B. glabrata (NMRI strain) snail (1 cm width) and place in a glass petri dish.
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Snails obtained from the 5th floor snail room - use net to obtain a snail from the tank and place in in a little bit of APW to carry down to the lab and use for the extraction.
3. Pour 70% ethanol onto the snail in the dish and wipe off the snail’s shell with a Kimwipe to remove any contaminants (this should also kill the snail).
4. Grab 100 mL beaker and press it down on top of the snail to crush it’s shell.
5. Using tweezers (and/or a dissection kit), dissect the snail from its shell. There will be a hard spot in the center - this is part of the shell and needs to be removed as well.
6. Use tweezers to transfer the dissected snail (no shell attached) to a 1.5 mL RNase/DNase-free tube.
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If extracting from multiple snails, or doing this procedure with a DNA extraction kit as well → label the top of the tubes with a marker!
7. Add 400 µL Trizol reagent to this tube and use a 1.5 mL tube pestle to begin homogenizing the snail tissue.
8. Cap this tube and drop into liquid nitrogen to freeze it. Then, remove the tube by grabbing it with tweezers.
9. Continue using pestle to homogenize the tissue.
10. Centrifuge this tube at 21,300 rcf. for 1 minute at room temperature.
11. Set a Zymo-Spin IICR Column inside one of the collection tubes from the RNA extraction kit and use a p100 to transfer the supernatant from the 1.5mL tube to the new spin column (~300 µL transferred)
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TIP: when transferring supernatant from the 1.5 mL tube, place the pipette tip just under the foam at the top and above the debris at the bottom of the tube (see image below)

12. Add an equal volume of 100% absolute ethanol (200 proof) to the spin column with the supernatant in it.
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TIP: estimate the volume and use a pipette set to your hypothesized volume of supernatant in the tube and test to make sure that is the correct volume. If it is, pipette it back into the tube and add that volume of ethanol. If not, use this same method to determine the actual volume before adding ethanol to the tube. The volume of the supernatant was ~325 µL and after adding ethanol, the total volume in the spin column was 650 µL (these numbers are just for reference - numbers depend on volumes pipetted)
13. Centrifuge the spin column/collection tube at 10,000 rcf. for 1 min. at room temperature. These centrifuge settings are used for the rest of the protocol.
14. Discard the flow-through liquid (in the bottom of collection tube) and place the spin column in a new collection tube.
1. Repeat steps 13 and 14 (centrifuge, discard flow-through, and replace collection tube) if not all of the liquid travelled through the column.
15. Add 400 µL RNA Wash Buffer to the spin column/collection tube and centrifuge. Discard flow-through and replace collection tube. Set this aside.
16. In an RNase-free tube, mix 5 µL DNase I with 75 µL DNA Digestion Buffer.
17. Add this solution (80 µL) to the spin column/collection tube and wait 15 minutes for the DNase I to digest the DNA in the sample.
18. After 15 minutes, add 400 µL Direct-zol RNA PreWash to the column and centrifuge. Discard flow-through, replace the collection tube, and repeat this step.
19. Add 700 µL RNA Wash Buffer to the spin column/collection tube and centrifuge.
20. Discard flow-through and replace the collection tube (carefully) with an RNase-free tube. The lid/cap will not be able to close - this is not a problem.
21. Add 50 µL of DNase/RNase-Free Water directly to the middle of the spin column matrix and centrifuge.
22. After centrifugation, obtain the eluate (flow-through now in the RNase-free tube) and discard the spin column. The extracted RNA is now in the eluate within the RNase-free tube.

Qualification of RNA Extract (NanoDrop Spectrophotometer)

1. Obtain your eluted sample from the RNA extraction protocol above and the eluent used (DNase/RNase-Free Water).
2. Turn on the NanoDrop Spectrophotometer and wipe both pedestals (one on the arm and one on the base) with a Kimwipe.

3. Pipette 1 µL of eluent (DNase/RNase-Free Water) onto the base pedestal and close the arm. Allow the screen to finish processing this before moving on to step 4.
4. Wipe off both pedestals with a Kimwipe and pipette 1 µL of the RNA sample onto the base pedestal.
5. Data will be generated (example image below). Make note of the values provided in the top right corner of the screen. [ng/µL, A260/A280, and A260/A230]

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INTERPRETATION: ng/µL tells you the concentration of nucleic acids in the sample. The NanoDrop isn’t the best machine to use for this. See below for a better method of quantification. The A260/A280 ratio tells you the amount of nucleic acids (which absorb at 260 nm) are in your sample relative to the amount of proteins (which absorb at 280 nm) in your sample. A value between 1.8-2.0 is considered pure. The 260/230 ratio tells you the amount of nucleic acids (260 nm absorbance) relative to the amount of organic compounds (230 nm absorbance). A value between 2.0-2.2 is considered pure, though 1.8-2.0 could be alright depending on the downstream experiments the sample will be used for. TROUBLESHOOTING: An A260/A280 ratio <1.8 usually indicates protein contamination. This might be an issue of incomplete lysis → try increasing the Trizol volume and ensure complete homogenization of sample. An A260/A230 ratio <1.8 most likely indicates Trizol, ethanol, or salt contamination. Try increasing the centrifuge time and/or speed to ensure all lysate and buffers are passing completely through the spin column. Wiping off the spin column with a Kimwipe between transfers to new collection tubes could also help.
6. Label RNA sample tube, freeze to store for later use or proceed with quantification of the sample.
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Label tube → “BG RNA” (BG → Biomphalaria glabrata), your initials, the date, and the concentration of the sample (ng/µL) Storage of Samples → Place in the PCR box in the fridge. It is a white box, labeled with blue tape saying → “B. glabrata (NMRI) DNA and RNA”

Quantification of RNA Extract (Invitrogen Qubit4 Fluorometer)

1. Determine your “n” value. This is equivalent to the number of samples you will be quantifying, plus one, plus 2 standards. For example, if you have 2 samples, the n value would be 5.
2. Load (n x 1 µL) to get the volume of Qubit RNA reagent to add to the working solution. Then, load (n x 199 µL) to determine the volume of Qubit RNA buffer to add to the working solution.
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Reagents are in a white box on the top shelf of bench 3 in P366. The buffers are next to this box. You may be able to use a 1.5 mL tube for the working solution or you may need a 15mL tube. It depends on the volumes that are needed.
3. Prepare 2 standards to be read. Add 190 µL of the working solution and 10µL of the standard solutions into Qubit assay tubes.
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Standards are found in the fridge. Make sure to grab the RNA Qubit pack, not the DNA Qubit pack.
4. Prepare your samples to be read. First, determine how much working solution to add to the Qubit assay tubes. It will be between 190-199 µL of working solution and 1-10 µL of your original sample. As a general rule, the more concentrated the RNA sample is, the lower volume of sample you will want to add and vise versa.
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For reference, an initial sample of ~44 ng/µL of snail RNA was extracted (according to the NanoDrop reading). It was determined that 2 µL of this sample would be added with 198 µL of the working solution to the Qubit assay tube to be read. If the concentration had been lower, say 4.80 ng/µL for example, perhaps 5 µL would have been added with 195 µL of working solution. Less concentrated = higher volumes of sample added. More concentrated = lower volumes of sample added.
5. Turn on the Invitrogen Qubit4 Fluorometer by tapping the screen. On the home screen, choose an assay type by selecting “RNA” in the bottom right corner (see image for reference).

6. Select “RNA Broad Range” from the list of assay types (image below for reference).

7. The first time you run an assay, you will see a blue “Read standards” button. If it isn’t the first time running an assay, the last date and time that standards were run will be shown at the bottom of the screen. You can read the standards again if necessary (this is recommended).
8. To enter standards, follow the on-screen prompts. The two standards should produce a standard curve with RFU (relative fluorescence units) values. The graph will display a positive linear relationship (there will be a straight line starting from the bottom left, stretching to top right of the graph). Make sure this is what is shown after reading the standards.
9. After the standard curve is generated, press the “Run Sample” button in the bottom right corner of the screen.
10. Input the volume of the original RNA sample added to the diluted tube. This volume was determined in step 4. Change the output units to “ng/µL”
11. Press the “Read tube” button at the bottom right of the screen.
12. The original concentration of RNA will be calculated. Record this value and write it on the original sample tube in place of the NanoDrop concentration.
13. Throw out the Qubit assay tubes and working solution. Store the original RNA extraction sample tube in the freezer box labeled, “B. glabrata (NMRI) DNA and RNA.”