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):

• T126-G

• T127-G

• T128-G

• T129-G

• T130-G

• T131-G

• T132-G

• T133-G

• T134-G

• T135-G

• T136-G

• T137-G

• T110-F_PG

• T111-F_PG

• T112-F_PG

• T46-F_PG

• T47-F_PG

• T49-F_PG

• T51-F_PG

• T52-F_PG

• T55-F_PG

• T56-F_PG

• T57-F_PG

• T58-F_PG

• T110-G

• T111-G

• T112-G

• T46-G

• T47-G

• T49-G

• T51-G

• T52-G

• T55-G

• T56-G

• T57-G

• T58-G

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 @ -80^oC in Sam’s RNA Box #2 and Sam’s RNA Box #3.

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RESULTS

Raw Qubit data (Google Sheet):

• 20220109_qubit_rna_mtro

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