Sam’s Notebook: RNA Isolation – Tanner Crab Hemolymph Pellet in RNAlater using TriReagent

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I previously isolated RNA from crab hemolymp from a lyophilized sample using TriReagent and Grace recently tried isolating RNA from crab hemolyph pellet (non-lyophilized) using TriReagent. The results for her extractions weren’t so great, so I’m giving it a shot with the following samples:

  • crab 424
  • crab 429
  • crab 438

Isolated RNA using TriReagent, according to manufacturer’s protocol:

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

Phase separation after chloroform addition was not particularly good. Aqueous phases in sample 424 was a bit cloudy (salty?) with no defined interphase. The remaining two samples did exhibit a defined interphase and were the aqueous phases were less cloudy than sample 424, but were far from ideal.

Quantified RNA using Roberts Lab Qubit 3.0 with the Qubit RNA high sensitivity kit. Used 5uL of each sample.

Grace’s Notebook: Plan for R and Bioanalyzer results

Today I discussed with Sam about how to use R for making a better master file. We identified some short term and long term goals for me to work on (and get help with via GitHub Issues). I also ran the Bioanalyzer on Test3 (from when lyophilizer was used and when ), and four samples that I extracted last Friday using (wihtout

R code goals

Long-term:

  • Create a code for making a hemolymph data sheet.

Currently, my hemo master data (20180522-all-crabs-hemo.csv) is copy and pasted from an excel file that Pam sent me. It’s copied from the tab labeled “all crabs”. The excel sheet has empty cells, so in order to make the .csv, I manually filled the cells.

Short-term:

  • Create code for reading in new Qubit files, creating a vector of the tube_numbers
  • figure out how to make the vector to join the Qubit data file (make it a column called “tube_number”)
  • add the “extraction_method” to the new Qubit file
  • add yields in ng
  • join the new Qubit file to the hemosample_qubit_table.csv based on “tube_number”

Having a workflow for this will make my life SO MUCH easier, as I have a lot more RNA extractions to do, and I have to keep track of which samples have been extracted, what the yields were, and extraction method.

Bioanalyzer results

Bioanalyzer file

I ran five samples in the Bioanalyzer.
Test 3, one of the lyophilized samples from what Sam extracted (the other two didn’t have detectable RNA in the Qubit, so Sam only gave me the sample with detectable RNA) using Tri-reagent protocol.
The other four samples are from what I extracted (not lyophilized) using Tri-reagent protocol on Friday.

Electropherogram:

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Gel:

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Thoughts on the results:
Clearly, the samples that I extracted on Friday were no good, even though they had detectable RNA.
The sample that Sam extracted looked quite good! So, it appears that lyophilizing samples before extracting using Tri-reagent is the way to go.

Now we just have to wait to hear when the lyophilizer is fixed… or think of something else to try in the meantime.

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