Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.
The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).
Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.
The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.
Samples were stored @ -80C. Will DNase next week.
The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):
Isolated RNA from 3 digestive gland (DG) samples: 08:13-7, 08:13-9, and 08:13-16 using the RNA Power Soil Kit (MoBio) according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.
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.
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.
Isolated RNA from Red Abalone Dg (10:25-24, 25, 26) using TriReagent, according to protocol. These samples are expected to have high levels of the Rickettsia phage that I’ve been attempting to investigate. The tissue samples were taken on 20110224 by Lisa and stored in RNA Later @ 4C. Remaining tissue will be returned to existing tubes and stored @ -80C. Previously, RNA from Ab Dg had been isolated using the MoBio RNA PowerSoil Kit due to its ability to produce extremely clean RNA from Dg (a nasty tissue). However, we currently do not have that kit. RNA was resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab ND1000. RNA will be stored in “Sam’s -80C Box #1″ in the Roberts’ Lab -80C freezer.
As expected, the RNA quality is low (based on OD260/280), as the TriReagent method of isolation does not clean up the RNA as extensively as the MoBio RNA PowerSoil Kit which has been previously used. Additionally, the RNA solution is brown colored. Will DNase RNA.
DNase – Abalone Dg RNA (from earlier today)
DNased Abalone Dg RNA that was isolated earlier today using Ambion’s Turbo DNA-free Kit following the “rigorous” protocol. Briefly, 5ug of RNA was brought up to 45uL with nuclease-free H2O. Reaction was brought to 50uL with 10x DNase Buffer and 1uL of DNase. Sample was incubated @ 37C for 30mins. An additional 1uL of DNase was added, mixed and incubated for 30mins. DNase was inactivated, supe transferred to clean tube and spec’d on Roberts’ Lab ND1000. DNased RNA will be stored in “Sam’s RNA Box #1″ in the Roberts’ Lab -80C freezer.
OD260/280 values still look poor (which was expected based on initial isolation from earlier today). Will check DNased RNA for residual gDNA carryover.