Kaitlyn’s notebook: Geoduck hemolymph next steps and RNA extractions

Recap

The overall goal is to identify whether the geoduck are reproductively developed (ready to spawn), and potentially whether the geoduck are male or female, using hemolymph (which can be non-lethally collected).

So far we have…

1.Identified biomarkers for reproductive development/sex

  1. Developed primers for biomarkers

Next Steps:

I want to test the newly developed primers, but first I need to ensure I have adequate amounts of RNA and cDNA because I now have 17 primer pairs to test.

Identified 9 female and 7 male hemolymph samples across stages for qPCR:

Sample Sex Stage
19 F 2
21 F 2
23 F 2
55 F 3
31 F 4
39 F 5
37 F 6
57 F 7
61 F 7
28 M 1
27 M 2
54 M 3
43 M 4
59 M 4
62 M 5
66 M 5

Samples for RNA extraction:

50ng RNA is needed for the RT protocol (100 ng is ideal esp. since I will use 1ul of template) so previous samples will need to be extracted again: 19, 21, 23, 55, 37, 28, 54, and 43.

Samples for RT (to make cDNA):

Samples that did not amplify previously may have had errors during RT so I will remake their RT in addition to the new samples: 19, 21, 23, 55, 37, 28, 54, 43, 39, 57, 62, and 66.


RNA Isolation:

RNA was isolated with a Quick-DNA/RNA Microprep Plus Kit by ZymoResearch according to the manufacturer’s protocol from geoduck hemolymph samples. 300ul of sample was used and 1200ul of lysis buffer was added for sample prep. All 250ul of sample 19 was used and 1000ul of lysis buffer added instead. The RNA was NOT DNased (will need to be done before RT).

I tested the ‘whole blood’ manufacturer instructions on sample 43B; sample 43A was done normally. Sample 54 was accidentally added to the sample 55 column. I pipetted out the supernatent from the 55 column, denoted the column as 55F, and added the supernatent to the correct 54 column. Then I got a new column for the second half of 55, labelled it as 55(2) and continued the extraction.

Samples were quantified with the hsRNA Assay for Qubit according to manufacturer’s protocol. 2ul of sample and 198ul of working solution was used per assay tube. Standard 1: 92.49 RFU and Standard 2: 1881.11 RFU.

Sample Sex Stage RNA (ng/ul)
19 F 2 35.8
21 F 2 18
23 F 2 low (151.89 RFU)
28 M 1 18.7
37 F 6 high (4893.75 RFU)
43A M 4 high (3520.34 RFU)
43B M 3 3.62
54 F 3 high (3152.78 RFU)
55F F 7 high (3544.7 RFU)
55(2) M 4 57

Although in RFU range, the low sample may be under ng/ul minimum and is likely not enough for RT. High samples will need to be diluted 1:2 and be requantified.

Samples are stored in a box in the -80C freezer in 3, 3, 2, labelled “RNA isolations; geoduck 12/17”. Note that blue tubes are from previous RNA isolations which were DNased, and those with ‘B’ denoted are from the second round of RNA extractions (contain 0ng RNA). The samples extracted today, which are not DNased, are in yellow tubes.

20200205_102703


How much cDNA should I make?

  • RT protocol provides 20ul of template
    • will use 1ul of template for qPCR
      • = total of 20 samples that can be done
        • run samples in duplicate…
          • must do 2 rounds of RT to get enough cDNA for 1 round of qPCR with 17 primers
  • RT calculations

I also picked up the new primers from Biochem stores which are on the benchtop in 209.

Yaamini’s Notebook: Gigas Broodstock RNA Extraction

C. gigas RNA and DNA extractions: Day 1

With everyone else in the lab extracting RNA, I figured I should too (#fomo…?). Over the next few days, I’ll extract RNA and DNA from frozen C. gigas tissues collected after 7 weeks of either low or ambient pH exposure in 2017. These are the adults I already extracted gonad DNA from.

I followed the ZymoResearch Quick DNA/RNA Microprep Plus Kit protocol, which is what Grace uses for her crab samples. Since Grace has been able to use it with her trickier crab hemolymph samples, I’m more confident that it should work with standard frozen oyster tissues. The kit separates out DNA and RNA into a column and flow-through, so I should be able to get both molecules from one sample. Today I’ll test out the protocol using adductor tissue samples from 20 individuals (10 low pH, 10 ambient pH). I decided to start with the adductor first since it’s the tissue I care about least. It’s not directly involved in any sort of ocean acidification acclimation processes, but could be affected. It might be a nice contrast to see how methylation, chromatin accessibility, and gene expression changes in relation to other tissues more active in ocean acidification acclimation like ctenidia and mantle tissues.

Methods: Sample Preparation

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.
  • Add 96 mL of 100% ethanol to the 24 mL DNA/RNA wash buffer concentrate.
  • Add 1040 µL Proteinase K Storage Buffer to Proteinase K vial. Vortex and store at -20ºC
  • Set heat block to 55ºC
  • Obtain samples from -80ºC freezer and place in ice: I did this before I did everything else since I was preoccupied with actually locating my samples. By the time I scrounged up the rest of my materials, they were more tissue than frozen…whoops.

Step 2: Cut and weigh no 0.005 g (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 tweezers to remove the tissue from the tube, then a razor blade to cut the tissue. Once I got the weight I wanted, I transferred the tissue to the labelled centrifuge tube with the tweezers.
  • Tweezers and the razor blade were washed in 200 mL of a 10% bleach solution, then in two separate DI water rinses. I wiped the tools clean with a kim wipe to use again.
  • I first tried using our lab’s scale with samples 18 and 19, but I couldn’t get a reading less than 10 mg even when barely any tissue. I borrowed the scale from Graham’s lab and was able to get actual weights.

Table 1. Mass of samples used for RNA extractions.

Sample ID Mass (mg)
1-T3 4.8
2-T1 4.7
3-T1 4.5
4-T3 5.1
5-T3 5.5
6-T1 5.3
7-T2 5.4
8-T2 5.1
9-T2 5.4
10-T3 5.3
11-T4 4.8
12-T6 4.4
13-T5 4.7
14-T6 4.8
15-T5 4.5
16-T4 5.5
17-T4 4.5
18-T6 4.6
19-T5 5.3
20-T6 5.0

Step 3: Add 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.

  • I intially added 300 µL DNA/RNA shield (2x) to each sample, but I only noticed this after the samples were on the heat block…I removed samples from the heat block at 3 p.m. and added 300 µL nuclease free water to dilute the DNA/RNA shield.

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

  • After adding 300 µL nuclease-free water, my sample volume had effectively doubled. I added an additional 30 µL PK Digestion Buffer and 15 µL Proteinase K, totalling 60 µL PK Digestion Buffer and 30 µL Proteinase K per sample.

Step 5: Place samples on a heat block at 55ºC for 2-5 hours.

  • Initially placed samples on the heat block at 1:55 p.m. At 3 p.m., I removed them from the heat block to correct mistakes in Step 3 and 4. I placed them back on the heat block at 3:20 p.m.

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

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

Methods: Sample Purification

Step 8: Transfer the sample into a IC-MX spin column in a collection tube and centrifuge at 15,000 x g for 30 seconds.

  • Keep the labelled spin columns for DNA extractions. I originally thought that I would extract RNA today and place the DNA column in the fridge for later extraction, but Sam told me that yields were really poor when he ketp samples in the fridge for a week. I decided to extract both nucleic acids today.
  • Save the flow-through for RNA extractions
  • Since I had to add extra liquid (s/o to past Yaamini for not internalizing directions), I had a lot of sample. Not all of the sample fit into the spin column. I spun the first 1000 µL (about 300 µL remaining) and processed the DNA columns first. When I removed the IC-MX columns with DNA from the collection tubes with flow-through and RNA, I completely spaced and forgot to label the collection tubes. By the time I went back to process them and elute RNA, I had no idea which sample was which! I had to discard the flow-through with RNA and tubes, but thankfully I still had sample remaining that had not gone through a collection tube. Good thing I’m making all these mistakes with low stakes samples that I have so much more of.

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 at 15,000 x g for 30 seconds. Discard the flow-through.

Step 10: Add 400 µL DNA/RNA Prep Buffer to the column. Centrifuge at 15,000 x g for 30 seconds. Discard the flow-through.

Step 11: Add 700 µL DNA/RNA Wash Buffer to the column. Centrifuge at 15,000 x g for 30 seconds. Discard the flow-through.

Step 12: Add 400 µL DNA/RNA Wash Buffer to the column. Centrifuge at 15,000 x g for 2 minutes. Transfer the column to a new microcentrifuge tube.

Step 13: Add 15 µL DNase/RNase-Free Water to the column. Incubate at room temperature for 5 minutes. Centrifuge at 15,000 x g for 30 seconds to elute the RNA.

Going forward

  1. Quantify yields from today
  2. Extract and quantify ctenidia RNA and DNA
  3. Extract and quantify mantle RNA and DNA

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