I finished RNA extraction protocol from the C. Gigas heat samples this week. Last week, I had extracted the RNA and was left with an RNA pellet, but I still had to complete the ethanol washes, so this week I finished the ethanol purification step and went to quantify the extracted RNA using the Qubit. The methodology I followed is described below:
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.
- Qubit assay was run, but RNA content in samples was beyond the limit of quantification for the machine. I will have to dilute the RNA samples and then re-quantify on Friday which should hopefully solve the problem.