Testing protocol for sperm samples

Now that I’m done with the female gonad samples, I need to extract DNA and RNA from sperm samples. According to this spreadsheet, the samples are in 100 µL of a seawater solution. I wasn’t sure if I should attempt to pellet the sample and remove the seawater. Based on Sam’s feedback this issue, I should proceed with the protocol for cells in suspension or other liquids. So that’s what I did!

Methods: Sample Preparation

Step 1: Prepare for extractions.

• Label 3 sets of tubes RNase-free centrifuge tubes per sample: one for the sperm sample, one for final RNA storage, and one for final DNA storage.
• Label spin columns
• Obtain samples from -80ºC freezer

Step 2: Obtain 50 µL of sperm sample in a seawater solution and pipet into a labelled centrifuge tube.

• I chose 50 µL of sample since I thought it would allow me to test the protocol without using all of the sperm sample sent
• After bringing the sample to room temperature, I pipetted up and down to mix and took out 50 µL. I noticed that there were small pieces of tissue in the solution, but it was mainly cloudy liquid. I assume this means that there may be some connective tissue in the samples.

Step 3: Add 4 volumes of DNA/RNA Lysis Buffer and mix

• I added 200 µL of Lysis Buffer to each sample for a total volume of 250 µL
  • For sample 12, the pipet tip got stuck and the liquid wouldn’t come out! I ended up using ethanol to clean a pair of scissors, then cutting off the pipet tip above where the liquid was to break the vacuum seal. I lost some liquid, but I had more than 200 µL of liquid in the tube. Since I mixed the buffer while adding it to my sample, I don’t think I only lost sample or buffer when I lost liquid.
• Vortexed for 10 pulses

Methods: Sample Purification

I also grabbed DNAse from the -20ºC freezer and put the nuclease free water on a heat block at 95ºC.

Step 4: Transfer the sample into a IC-MX spin column in a collection tube and centrifuge.

• Kept the labelled spin columns for DNA extractions
• Saved the flow-through for RNA extractions

Step 5: For RNA only, add an equal volume of 95-100% ethanol to the flow-through and mix by pipetting. Transfer the flow-through into a new IC spin column in a clean collection tube. Centrifuge and discard the flow-through.

• Grace took on the RNA tubes!
  • Added 250 µL of EtOH and mixed

Step 6: For the RNA IC spin columns, perform a DNAse I treatment

• Grace did this!
  • Added 400 µL of the DNA/RNA Wash Buffer to each column and centrifuged, then discarded the flow-through.
  • Made a DNase REaction Mix with 5 µL of DNase I and 35 µL DNA Digestion Buffer for each sample.
  • Added the mix to the column matrix and incubated it at room temperature for 15 minutes before proceeding.

Step 7: Add 400 µL DNA/RNA Prep Buffer to the column. Centrifuge and discard the flow-through.

Step 8: Add 700 µL DNA/RNA Wash Buffer to the column. Centrifuge and discard the flow-through.

Step 9: Add 400 µL DNA/RNA Wash Buffer to the column. Centrifuge at 16,000 rcf for 2 minutes. Transfer the column to a new microcentrifuge tube.

Step 10: Add 15 µL DNase/RNase-Free Water to the column. Incubate at room temperature for 5 minutes. Centrifuge to elute the DNA and RNA.

• Added 15 µL of 95ºC nuclease-free water to the column
• Samples were centrifuged for 1 minute

Methods: Sample Quantification

Step 11: Prepare the master solution using a 1:200 ratio of buffer to dye. For each sample and both standards, 200 µL of master solution is needed.

• RNA: Grace did this!
• DNA: Pre-mixed master solution for the Qubit dsDNA HS kit (aka my favorite thing)

Step 12: For each standard’s designated Qubit assay tube, add 10 µL of the correct standard and 190 µL of the master solution.

Step 13: For each sample’s designated Qubit assay tube, add 1 µL of the sample and 199 µL of the master solution.

Step 14: Vortex all Qubit assay tube for 2-3 seconds at maximum speed. Incubate tubes at room temperature for two minutes.

Step 15: Use Qubit to quantify RNA and DNA yield in each sample tube.

• DNA: S1 = 80.03, S2 = 39903.70; RNA: S1 = 83.23, S2 = 1864.03
• Updated yields can be found here
• I’m worried that I won’t have enough RNA for RNA-Seq libraries with 50 µL of starting volume. It’s time to get some quotes and figure out how much RNA is needed at different facilities. I could also use the remaining 50µL of each sample for RNA exractions, but we’ll see.

Going forward

1. Extract male samples and check yields
2. Send DNA and RNA for library preparation and sequencing