RNA Isolation - M.trossulus Gill and Phenol Gland

2022
Miscellaneous
Author

Sam White

Published

January 9, 2022

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA was isolated using RNAzol RT and the Direct-zol RNA Microprep Kit (ZymoResearch), with the DNase I on-column treatment step. Foot tissues were allowed to partially thaw to allow me to unfold/unbend the foot and dissect the phenol gland (essentially the most distal end of the foot) with a new razor blade. For gill tissue, small portions of frozen gill tissue were removed from tubes, placed in RNAzol RT and immediately homoegnized. Forceps were rinsed in distilled H2O, soaked in 10% bleach solution for 10mins, and then rinsed in distilled H2O before re-use. All centrifugation steps were performed at 16,000g for 1.5mins. Here’s a brief overview of the process.

Tissues were homogenized in 500uL of TriReagent with “disposable” platic mortar/pestle tubes (1.5mL). After homogenization, an additional 500uL of TriReagent was added to the tube, vortexed and incubated at RT for 10mins. Insoluble debris was pelleted and supernatant was transferred to a 2.0mL tube. An equal volume (1mL) of 100% ethanol was added to this supernatant and mixed thoroughly by pipetting. Direct-zol Microprep Kit protocol was followed from here on, including on-column DNase I treatment. All samples were eluted with 100uL of H2O.

RNA was quantified using the Roberts Lab Qubit 3.0 using the Qubit RNA High Sensitivity assay.

All RNA was stored @ -80oC in Sam’s RNA Box #2 and Sam’s RNA Box #3.

RESULTS

Raw Qubit data (Google Sheet):

Virtually all isolations looked good, except T47-F_PG, which had an extremely low yield (only 278ng). Not sure what went wrong here, as I don’t recall have any specific difficulties with the isolation (e.g. clogged column). Will keep this sample in mind and will likely return to it at a later date, but will let that decision belong to Matt George after he makes decisions regarding sequencing for this project.

Summary Table:

sample tissue concentration(ng/uL) volume(uL) yield(ng)
T126-G gill 79 100 7900
T127-G gill 86.6 100 8660
T128-G gill 82 100 8200
T129-G gill 45.8 100 4580
T130-G gill 62.6 100 6260
T131-G gill 31 100 3100
T132-G gill 94.8 100 9480
T133-G gill 56 100 5600
T134-G gill 32.6 100 3260
T135-G gill 44.2 100 4420
T136-G gill 48.6 100 4860
T137-G gill 20.2 100 2020
T110-F_PG phenol gland 35.6 100 3560
T111-F_PG phenol gland 29.8 100 2980
T112-F_PG phenol gland 43.6 100 4360
T46-F_PG phenol gland 21.8 100 2180
T47-F_PG phenol gland 2.78 100 278
T49-F_PG phenol gland 17.4 100 1740
T51-F_PG phenol gland 124 100 12400
T52-F_PG phenol gland 37 100 3700
T55-F_PG phenol gland 32.6 100 3260
T56-F_PG phenol gland 64.4 100 6440
T57-F_PG phenol gland 91.2 100 9120
T58-F_PG phenol gland 28 100 2800
T110-G gill 42.2 100 4220
T111-G gill 40.6 100 4060
T112-G gill 24.2 100 2420
T46-G gill 19.8 100 1980
T47-G gill 47.8 100 4780
T49-G gill 56.6 100 5660
T51-G gill 8.48 100 848
T52-G gill 57.6 100 5760
T55-G gill 56.2 100 5620
T56-G gill 102 100 10200
T57-G gill 112 100 11200
T58-G gill 96.4 100 9640