Extract RNA from non-lyophilized C. bairdi hemolymph

Today I extracted RNA from four practice samples using Tri-reagent, and without lyophilizing (lyophilizer is out of commission again for now).

Tubes I used:

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101-A –> a mistake sample
471-1 –> Day 26, uninfected, ambient
462-2 –> Day 26, infected, ambient
482-2 –> Day 26, infected, ambient

Protocol (from Tri-reagent protocol in Sam’s notebook)

  1. Added 1mL TriReagent to each tube
  2. vortexed to mix/dissolve solute
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  3. incubated 5mins at RT
  4. added 200uL of chloroform, pipet and then vortexed 15s to mix
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  5. incubated at RT for 5mins
  6. centrifuged 15mins, 12,000g, 4˚C
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  7. transferred aqueous phase to new tube
  8. added 500uL isopropanol to aqueous phase
  9. mixed, incubated at RT for 10mins
  10. centrifuged 8mins, 12,000g, at RT
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  11. discarded supernatant
  12. added 1mL 75% ethanol
  13. centrifuged 5mins, 12,000g at RT
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  14. discarded supernatant
  15. resuspended in 10uL of 0.1% DEPC-treated H2O.

Qubit Results (5ul of sample):
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The extracted RNA tubes live in Rack 5, Column 4, Row 3

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I did not do anything with the supernatant. I only process hemolymph pellet samples today.

Thoughts on the protocol:

  • At step 14, tube 482 was really difficult to discard the supernatant. THere was stringy, clear material that was difficult to avoid, so I wasn’t able to discard a lot of the 75% ethanol. In the other tubes, however, the stringy stuff was more or less easier to avoid and I was able to remove almost all of the 75% ethanol before resuspending the sample in 0.1% DEPC-treated H20.
  • The aqeous phase was really apparent with the Trizol, which I never saw when I used the RNAzol. Using the RNAzol always ended up being that the blue RNAzol was either at the top of the tube, the middle/bottom, or sometimes even throughout the tube. This, in all four tubes, created a clearly defined aqeous phase, which made removing the aqeous phase so so easy.
  • Even though the Qubit results were pretty good, as I have learned from my RNAzol situation this summer, I cannot trust it until I look at the samples on the Bioanalyzer!!
Written on October 5, 2018