On 11/8, I finished up the RNA extraction for the final 16 samples of the desiccation + elevated temp. exposure (D05, D06, D07, D08, D15, D16, D17, D18, T05, T06, T07, T08, T15, T17, T18). I also ran the Qubit assay for 24 samples (D03, D04, D05, D06, D07, D08, D13, D14, D15, D16, D17, D18, T03, T04, T05, T06, T07, T08, T13, T14, T15, T16, T17, T18). 4 samples (D06, T05, T15, T17) had RNA concentrations above the limit of quantification, so I will have to dilute those samples and re-run the Qubit assay. Described below is the protocol for both the RNA extraction wrap-up and the Qubit assay:
RNA Extraction Wrap-Up:
- Stored RNA pellets (suspended in ethanol) were taken out from the -80 freezer and left to thaw for around 10 minutes.
- Supernatant was removed from all samples and 400 μL of 75% ethanol was subsequently added to each sample.
- Each sample was then centrifuged for 5 minutes at 1200 g.
- Supernatant was once again removed from each sample and each sample was then microcentrifuged for approximately 10 seconds so that any residual ethanol could be removed.
- 50 μL of DEPC water was added to each sample and samples were then vortexed to dissolve the RNA pellet. One sample’s (D11) RNA pellet did not fully dissolve, so an additional 50 μL of DEPC water was added and pellet was manually broken up by vigorously pipetting.
RNA Quantification (Qubit)
- 3980 μL of Qubit buffer and 20 μL of Qubit dye were added to a tube to create a 200:1 ratio between buffer and dye.
- 198 μL of the mastermix and 2 μL of the RNA samples were added to each of 16 Qubit tubes (1 tube for each sample).
- 2 standardization tubes were also set up. 190 μL of mastermix and 10 μL of Qubit standard #1/2 were added to 2 Qubit tubes.