INTRO

Continued with co-isolation of DNA and RNA from Pacific cod blood samples selected by Laura (GitHub Issue).

MATERIALS & METHODS

HOMOGENIZATION

Blood samples were thawed on ice and then 200uL of 2x DNA/RNA Shield was added to each and vortexed. The liquid and the gelatinous blood clot were transferred to 1.5mL SafeLock snap cap tubes (Eppendorf). I then added beads in the recommended proportions for the Bullet Blender Fresh Blood protocol. Samples were subjected to two rounds of “blending,” based on sample appearance. Blending was performed in a 4C cold room.

Here is a brief overview of the protocol:

Bead combination:

0.5 mm zirconium oxide beads (ZROB05). Use a volume of beads equivalent to 0.25 x the volume of the sample plus
0.15 mm zirconium oxide beads (ZROB015). Use a volume of beads equivalent to 0.25 x the volume of the sample

Procedure

1.Place the sample in the tube with the beads. 2. Close the tubes tightly and place them in the Bullet Blender. 3. Set the controls for Speed 10 and Time 3. Press Start. 4. After the run, remove the tubes from the instrument and visually inspect the samples. If homogenization is incomplete, repeat the homogenization step. 5.Proceed with your downstream application.

DNA/RNA Isolation

DNA and RNA isolations were performed using the Quick-DNA/RNA MiniPrep Plus Kit (ZymoResearch).

Note

Protocol was followed with the foll# SUMMARYowing notes/changes:

Did not perform Proteinase K digestion after homogenization.
RNA was treated with DNase I.
DNA was eluted with 100uL of H₂O.
RNA was eluted with 50uL of H₂O.

Quantification

DNA was quantified using the Roberts Lab Qubit 3.0 and the 1x High Sensitivity dsDNA Assay (Invitrogen), using 1uL of each sample.

RNA was quantified using Roberts Lab Qubit 3.0 and the RNA High Sensitivity Assay (Invitrogen), using 1uL of each sample.

RESULTS

SUMMARY

DNA

Qubit (Google Sheet):

20250410-qubit-gmac-blood-brDNA

sample_ID	Concentration (ng/uL)	Volume (uL)	Yield (ng)
91B	12.4	90	1116
92B	24.8	90	2232
94B	4.4	90	396
95B	18.3	90	1647
96B	24	90	2160
97B	5.26	90	473.4
98B	8.86	90	797.4
99B	16.2	90	1458
100B	5.12	90	460.8
101B	4.6	90	414
102B	9.32	90	838.8
103B	5.9	90	531

RNA

Qubit (Google Sheet):

20250410-qubit-gmac-blood-hsRNA

sample_ID	Concentration (ng/uL)	Volume (uL)	Yield (ng)
91B	26.2	40	1048
92B	66.4	40	2656
94B	2.04	40	81.6
95B	43	40	1720
96B	57	40	2280
97B	6.38	40	255.2
98B	4.14	40	165.6
99B	57.2	40	2288
100B	5.2	40	208
101B	59	40	2360
102B	8	40	320
103B	24	40	960

SUMMARY

DNA yields weren’t as good as I would’ve expected hoped, but most are probably sufficient. RNA yields were mostly good and are suffiient for RNA-seq