Oly gonad & larvae RNA isolation
Gonad RNA isolation:
Olympia oyster gonad samples were fixed in PAXgene tissue fixative/stabilizer in April, 2017, directly after removing them from pH treatments (~7.2, ~7.8 for 8wks; prior to the pH treatments animals had been in temperature treatments, 6C & 10C for 8wks). Sam previously extracted RNA from 12 of my gonad samples, 6 of which were from the South Sound F1 groups. Today, I extracted (most of) the remaining South Sound gonad samples.
First, I used a razor blade to dig tissue out of the paraffin blocks, using the corresponding slides to identify and target only gonad area for my tissue sections.
For RNA extraction, I used the same kit - PAXgene fixed tissue RNA kit, following the instructions for for tissue in histology blocks. I followed the kit procedure, with the following alterations:
- To homogenize: Added 1 spoonful of glass beads (autoclaved) to each sample, and vortexed for ~45 minutes on the vortexer speed 9 with the foam tube holder attachment. At minute ~25, moved samples from the inside ring to the outside ring.
- Max centrifuge speed for steps 4, 7 & 12 = 13rpm
- Max centrifuge speed for steps 22-24 = 15rpm
- Final elution volumes: 1st elution of 20ul, 2nd with 30ul, for total volume ~48-50ul (kit says I will lose ~2ul to the column).
- I began extracting 30 samples, but then found that I only had 26 columns, so I terminated 4 samples, one from each treatment.
- Sample #23: I ran out of the DNase + Buffer mixture for this final sample at step 18, I only had ~1/4 of the required mix. Because I had no more volumes of DNase left, but noticed a few small droplets on the walls of a DNase tube, I added 20ul of buffer to the DNase tube, flicked to mix, then centrifuged for 2 seconds and added that (~20-ish ul) to sample 26.
Quantified RNA using the Qubit RNA assay with Qubit 3.0 Fluorometer. Used 2.0ul of each sample for quantification. Approximate RNA (ng) is calculated using concentration (ng/ul) in 46ul.
|RNA Sample #||Sample #||pH||Tissue Type||Sample date||Extraction Date||RNA Concentration (ng/ul)||Approximate ng RNA (in ~46ul)||Notes|
|x||SN-6-19||Low||Fixed Gonad||4/8/17||Did not fully extract (ran out of columns)|
|x||SN-6-31||Ambient||Fixed Gonad||4/8/17||Did not fully extract (ran out of columns)|
|22||SN-10-22||Low||Fixed Gonad||4/8/17||Did not fully extract (ran out of columns)|
|SN-10-25||Ambient||Fixed Gonad||4/8/17||7/?/2017||Did not do - already sent to Katherine|
|SN-10-26||Ambient||Fixed Gonad||4/8/17||7/?/2017||Did not do - already sent to Katherine|
|SN-10-30||Ambient||Fixed Gonad||4/8/17||Did not fully extract (ran out of columns)|
|SN-10-31||Ambient||Fixed Gonad||4/8/17||7/?/2017||Did not do - already sent to Katherine|
|32||SN-10-32||Ambient||Fixed Gonad||4/8/17||3/27/18||Out of range - too high||#VALUE!|
Spreadsheet with RNA sample key and ng isolated is in the O.lurida_Stress repo