Grace’s Notebook: Bairdi hemolymph extraction plan (work in progress)

Here’s the general extraction plan. There will be a final version of this extraction plan of actually selecting the tube numbers, and taking into account whatever feedback I get.

Number of samples to process

Based on previous RNeasy processes (Feb 15th, and Feb 20th), we have decided that using 50ul or less of the hemo slurry is best with the RNeasy Kit.

I’ll use 20ul of the hemolymph slurry from about 150 individual sample tubes. (Number of tubes processed will depend on the RNA yield we get)

I’ll process 8 samples at a time, and will select tube numbers such that each infection status and temperature treatment group has 8 samples selected, along with 4 back-up tubes in case there isn’t detectable RNA in any of the samples.

To start, we’ll do 12 sets of 8 tubes, running 1ul of each sample on the Qubit at the end of each set, keeping track of which pools need extra RNA. Then, after the 12 sets of 8, I’ll collect whatever extra tubes we need from the back up list that I’ll make and decide how many additional sets need to be processed.

The goal is to get to the point where I have 12 pooled samples, each with at least 1000ng RNA in 50ul of DEPC-treated H20.

Protocol preparation

Q: Should I prep huge containers of reagents or should I prep them fresh before each extraction?

Make:
70% ethanol
80% ethanol
Buffer RLT plus 2-BME

RNeasy

  1. Add 350ul of Buffer RLT Plus + B-ME solution to both samples
  2. Vortex to mix
  3. Transfer lysate to QIA Shredder column with 2ml collection tube. Centrifuge 2min at full speed
  4. Transfer flow-through to gDNA Elimninator column with 2ml collection tube. Centrifuge for 30s at 12,00 g. Discard column. Save flow-through. Also save gDNA column for later use.
  5. I measured the amount of flow-through for both samples and added that same volume’s worth of 70% ethanol. THe 10ul starting material sample had 340ul, so I added 340ul of 70% ehtanol. The 50ul sample had 345ul, so I added 345ul of 70% ehtanol. Mix by pipetting.
  6. Transfer sample (including any precipitate that may have formed) to RNeasy MinElute column. Close Lid. Centrifuge 20s at 12,000g. Discard flow-through
  7. Add 700ul Buffer RW1 to RNeasy column. Close lid. centrifuge 20s at 12,000g. Discard flow-through.
  8. 500ul Buffer RPE to RNeasy column. Close lid. Centrifuge 20s at 12,000g. Discard flow-through.
  9. Add 500ul 80% ethanol to RNeasy column. Close lid. Centrifuge 2min at 12,000g. Dsicard collection tube and flow-through.
  10. Put RNeasy column in new 2ml collection tube. Cut off RNeasy column lid. Keep tube open and centrifuge at full speed for 5min. Discard flow-through.
  11. Put RNeasy column in new 1.5 ml collection tube. Add 14ul RNase-free water (from red-capped aliquotted tube) to center of membrane (I missed the center for the 50ul sample). I forgot to close lid, so the tubes were centrifuged open for 1min at full speed.

Qubit
Run 1ul of each sample tube on the Qubit using RNA High Sensitivity
KEEP TRACK OF THE TUBE NUMBERS

Upload the Qubit results from each set of 8 immediately to my laptop, add the tube numbers manually, and save.

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