Tweaking Zymo MicroPrep Kit protocols

Based on my yields Friday, 5 mg of tissue definitely gets me enough RNA but not enough DNA. I wanted to see if increasing my starting tissue mass would increase my DNA yield without clogging the spin columns!

Methods: Sample Preparation

I started this process (Steps 1-5) Sunday Oct. 25 so I could conduct an overnight incubation.

Step 1: Prepare for extractions.

• Label 3 sets of tubes RNase-free centrifuge tubes per sample: one for frozen tissue, one for final RNA storage, and one for final DNA storage.
  • I labelled the tubes “36-P DNA” or “54-P RNA” to differentiate between “36 DNA” or “54 RNA”
• Set heat block to 55ºC
• Obtained tissue samples from -80ºC freezer and place on wet ice

Step 2: Cut and weigh no more than 5 mg of frozen tissue. Record weight of tissue used in extractions and place tissue in a new, labelled test tube.

• I tared the scale with a piece of weigh paper. I used a clean spatula with a sharp edge to cut the tissue in the tube. Once I got the weight I wanted, I transferred the tissue to the labelled centrifuge tube with the tweezers.
• I only had 7.7 mg left in sample 36, so I decided to use all of it and keep the reagents in the sample tube sent by Alan. Sample 54 had more tissue, so I could use ~10 mg.
• I smushed the sample with the spatula to help break it down.

Table 1. Mass of samples used for DNA/RNA extractions.

Sample ID	Mass (mg)
36-P	7.7
54-P	12

Step 3: Added at least 150 µL of DNA/RNA shield (2X) and 150 µL nuclease-free water to create 300 µL DNA/RNA shield (1x) to each sample. If the sample is not covered by water, add more DNA/RNA shield (1x) until covered and record the volume of liquid added.

• At this step, I also vortexted the samples to help break them down before I added more reagents. 36 was more broken down than 54.

Step 4: For every 300 µL of sample, add 30 µL PK Digestion Buffer and 15 µL Proteinase K. Mix by vortexing gently.

Step 5: Place samples on a heat block at 55ºC overnight.

• I started the incubation at 6:50 p.m.

Methods: Sample Preparation

Before I stopped the sample incubation, I added 1040 µL of Proteinase K Storage Buffer to a new tube of Proteinase K, since I almost ran out last night. I also located a reconsituted DNase I in the -20ºC (bottom rack, miscellaneous enzymes box) and brought it to room temperature for the DNase I treatment.

Step 6: Vortexed sample and centrifuge at maximum speed for 2 minutes to pellet debris. Transfer the aqueous supernatant to an RNase-free tube.

• I removed the samples from the heat block at 10:30 a.m. I aliquotted 1000 µL of nuclease-free water, put it on the heat block, and set it to 95ºC.
• To vortex, I did 10 pulses at maximum speed
• To centrifuge the samples, I needed to transfer the contents of the sample 36 tube to a labelled centrifuge tube. During this process, I lost some sample because I had previously vortexed it and it was very bubbly. I did my best to suck up the bubbles into the pipet tip, but there was definitely some liquid I couldn’t get!
• Samples were centrifuged at 21,130 rcf

Step 7: Add an equivalent volume of DNA/RNA Lysis Buffer to each sample and mix by vortexing.

• Added 345 µL of DNA/RNA Lysis Buffer to each sample

Methods: Sample Purification

Step 8: 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 9: 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.

• Added 750 µL of EtOH and mixed. Had to centrifuge twice because the column could only hold 750 µL of liquid at once, so the original volume was greater than 750 µL

Step 10: For the RNA IC spin columns, perform a DNAse I treatment

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

Step 11: Add 400 µL DNA/RNA Prep Buffer to the column. Centrifuge and discard the flow-through.

Step 12: Add 700 µL DNA/RNA Wash Buffer to the column. Centrifuge and discard the flow-through.

Step 13: 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 14: 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
• We did a 30 second elution, but I ran sample 36-P DNA twice since the final elution volume looked like less than 15 µL the first time.

Methods: Sample Quantification

Step 15: 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 made the master mix solution since she’s done more RNA HS Qubit runs
• DNA: Pre-mixed master solution for the Qubit dsDNA HS kit (aka my favorite thing)

Step 16: For each standard’s designated Qubit assay tube, add 10 µL of the correct standard and 190 µL of the master solution.

Step 17: For each sample’s designated Qubit assay tube, add 1 µL of the sample and 199 µL of the master solution.

Step 19: Vortex all Qubit assay tube for 2-3 seconds at maximum speed. Incubate tubes at room temperature for two minutes.

Step 20: Use Qubit to quantify RNA and DNA yield in each sample tube.

• DNA: Sample 36 had too high of a concentration! I tried a 1:10 dilution but that ended up being too low, so 1:5 was the Goldilocks dilution that lead to a Qubit reading
• RNA: Both samples were too concentrated! A 1:10 dilution worked for 54-P RNA, but we needed a 1:20 dilution for 36-P DNA. It makes sense that the high-yield sample is consistent between DNA and RNA.
• Final sample volumes after Qubit quantification: 14: 54-P DNA, 13: 54-P RNA, 12: 36-P DNA and RNA

Results

Table 2. DNA and RNA concentration for extracted samples. A total of 15 µL of RNA or DNA was eluted per sample. No dilution was used for 54-P DNA, a 1:5 dilution was used for 36-P DNA, a 1:10 dilution was used for 54-P RNA, and a 1:20 dilution was used for 36-P RNA. DNA S1: 71.54, DNA S2: 37192.48, RNA S1: 80.34, RNA S2: 15468.87

Sample ID	DNA Concentration (ng/µL)	DNA Yield (µL)	Total DNA Yield (ng)	RNA Concentration (ng/µL)	RNA Yield (µL)	Total RNA Yield (ng)
36-P	30.8	12	1848	166	12	39840
54-P	58.2	14	814.8	78.2	13	10166

Yield looks great! I think 10 mg, manual homogenization, and warmed nuclease-free water for elution is the way to go…at least for the female samples. Since the males are in seawater, I’ll need to re-evaluate the protocol for those samples. For now, I’ll continue with the female gonadal tissue!

Going forward

1. Subsample 10 mg of tissue for each female sample
2. Perform DNA and RNA extractions for the remaining female samples
3. Check DNA and RNA yield and quality for female samples
4. Test extraction protocol for male samples
5. Send DNA and RNA for library preparation and sequencing