INTRO

Steven tasked me with helping and/or isolating RNA (GitHub Issue) for Valentina (visiting scientist from Cristian’s lab in Chile). Samples were whole body (no shells) of small juvenile oysters exposed to poly I:C (Wikipedia) and then subjected to mechanical stress, or no mechanical stress.

MATERIALS & METHODS

Whole body tissue had been snap frozen and stored at -80^oC.

Homogenization

300uL of DNA/RNA Shield (ZymoResearch) were added to the frozen tissue. Samples and DNA/RNA Shield were transferred to 1.5mL SafeLock (Eppendorf) microcentrifuge tubes containig ~50uL of 0.5mm zirconium oxide beads and ~50uL of 0.15mm zirconium oxide beads. Samples were homogenized using the Roberts Lab Bullet Blender 5E Gold + (NextAdvance) with 1.5mL tube adapters for 3mins at speed 12. The Bullet Blender was pre-cooled, and cooled, using dry ice.

RNA Isolation

RNA was isolated using the Quick-DNA/RNA MiniPrep Plus Kit, according to the manufacturer’s protocol with the following changes/notes:

Note

Treated with optional DNase step.
Eluted with 50uL of H₂O.

RNA Quantificaiton

RNA was quantified using the Qubit High Sensitivity RNA Assay (hsRNA), using 1uL of each sample.

The initial round of quantification revealed that most samples were too concentrated, so a 1:10 dilution of those samples were prepared and quantified.

RESULTS

Qubit data

20250804-cgig-hsRNA-polyIC (Google Sheet)

Sample	Concentration(ng/uL)	Dilution Factor	Actual Concentration (ng/uL)	Volume (uL)	Yield (ng)
A1 P C	26.4	10	264	40	10560
A2 P C	142	1	142	40	5680
A3 P C	112	1	112	40	4480
A4 P C	26.8	10	268	40	10720
A5 P C	150	1	150	40	6000
B1 P C	31.4	10	314	40	12560
B2 P C	34.8	10	348	40	13920
B3 P C	196	1	196	40	7840
B4 P C	190	1	190	40	7600
B5 P C	84.6	1	84.6	40	3384
A1 P M	23.4	10	234	40	9360
A2 P M	52.8	10	528	40	21120
A3 P M	27.4	10	274	40	10960
A4 P M	37.2	10	372	40	14880
A5 P M	28.8	10	288	40	11520
B1 P M	63	1	63	40	2520
B2 P M	35.2	10	352	40	14080
B3 P M	28.8	10	288	40	11520
B4 P M	200	1	200	40	8000
B5 P M	42.4	10	424	40	16960