Isolated RNA from 11 Olympia (O. lurida) oyster larvae samples, from 4 different pCO2 treatments using TriReagent (MRC) according to the manufacturer’s protocol. RNA was resuspended in 100uL of 0.1% DEPC-H2O, spec’d on the Roberts Lab NanoDrop 1000, and stored @ -80C.
Isolated RNA from the fractions collected on 20120525 during the differential centrifugation procedure: Gradient Top, Gradient Junk, Gradient Bottom, Hot PE Pellet, 12:6-1 (Control PE). Samples were isolated with in 1mL TriReagent according to protocol. Samples were resuspended in 0.1% DEPC-H2O, spec’d on the Roberts’ Lab NanoDrop1000 and stored @ -80C.
The second “Gradient Junk” sample on the spec results above is actually the control 12:6-1 sample.
Overall, the sample quality (based on the OD260/280) looks poor. However, this is NOT uncommon for this tissue type (PE gland) from abalone. Yields from the control sample (12:6-1) and the Hot PE Pellet are excellent. The yields from the gradient samples are low and won’t provide enough sample for high-throughput sequencing (for which this procedure was performed). Will discuss with Steven, Carolyn and Lisa for how we want to proceed.
Post-esphagus (PE) tissue was isolated from one control abalone (12:6-1; 0.077g PE) and three “hot” abalone (11:8-8, -9, -10; 0.1606g PE, 0.126g PE, 0.1205g PE) by Lisa. Control abalone PE was homogenized in 0.5mL TriReagent and stored @ -80C. The three “hot” abalone PE were individually homogenized in ice cold 5mL of 1x Tris Sucrose Buffer (TSB). pH = 7.4 until the entire tissue was fully homogenized, including the difficult connective tissue.
Samples were transferred to 15mL conical tubes and spun at 250g for 10mins @ 4C. The supe was transferred to a 30% Percoll-TSB gradient. The pellet was placed into 1mL TriReagent, vortexed and stored @ -80C (Hot PE Pellet). 25uL from the pellet and the supe were saved for qPCR analysis.
The gradient was spun at 25000g for 2hrs at 4C in a Sorvall T21 centrifuge in a SL-50T rotor with the “SoftSpin” setting on and the brake turned off.
Below is a link to a slide show of the sample at various stages of preparation, including images of what the gradient looked like after the final spin.
Isolated RNA from pinto abalone larvae that were sampled on 20110930. Samples were provided by Liza.
Due to large volumes of sea water in each sample (ranging from 500uL to 2000uL), samples had to be thawed entirely and then as much sea water was removed as physically possible. Samples were homogenized in 500uL of TriReagent (Molecular Research Center). An additional 200uL of TriReagent was added to each sample after homogenization to bring the final sample volume to ~1000uL. Samples were then processed according to the manufacturer’s protocol. Samples were resuspended in 50uL of 0.1% DEPC-treated H2O, heated at 55C for 10mins to coax the RNA into solution and spec’d.
Yields seem excellent (~80ug of total RNA per sample) as do the OD260/280 ratios. RNA will be stored @ -80C (will pass samples to Liza to decide on the physical location in the -80C) until a decision is made on what to do with the samples next.