INTRO

Steven and Laura decided to move forward with isolating DNA and RNA from Pacific cod blood samples (GitHub Issue) and tasked me with extractions. Between waiting on supplies and being out of town, I finally was able to begin tacklng the samples.

The full list of blood samples can be found here:

Cod blood samples for DNA & RNA (Google Sheet; restricted viewing)

SAMPLE SELECTION

Laura noted that samples 1-36 are the most precious. Based on the initial attempts at co-isolating DNA/RNA from cod blood on 2024-08-29, I decided to avoid those samples, since I was not entirely confident about this protocol just yet. See the RESULTS section for list of samples processed.

MATERIALS & METHODS

DNA & RNA Isolations

Important

Gah! After this was completed I realized I f’ed up the Proteinase K digestion! I didn’t realize the Proteinase K did not come in solution. That means I simply added Proteinase K buffer at the digestion step, instead of Proteinase K. I have since reconstituted and aliquoted the Proteinase K. Ugh.

I used the Quick-DNA/RNA MiniPrep Plus Kit (ZymoResearch) (PDF; GitHub) to isolate DNA and RNA from Pacific cod samples (see RESULTS for list of samples), according to the manufacturer’s protocol for whole blood, with the following modifications:

After the initial addition of isopropanol to the samples (Step 3), I pelleted unlysed cells/debris by spinning 30s at 16,000g to prevent subsequent clogging of the columns. The resulting supernatant was transferred to the columns.
The optional DNase step was used in the RNA isolation protocol.
Both DNA and RNA were each eluted with 50uL of H₂O.

Note

Due to heavy coagulation, after the initial addition of DNA/RNA Shield, I attempted to physically disrupt the coagulated blood using disposable pestles. However, this did not seem to have much of an effect.

Quantification

DNA was quantified using the Qubit 1x dsDNA High Sensitivity Assay (Invitrogen). 1uL of each DNA sample was used.

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

DNA and RNA were both stored @ -80^oC in the box: P.cod Blood DNA/RNA #1. This has been temporarily stored in the middle -80^oC freezer until space becomes available in the Roberts Lab -80^oC freezer.

RESULTS

DNA Quantification

20250325-gmac-qubit-DNA.hs-blood (Google Sheet)

Sample	Concentration (ng/uL)	Volume (uL)	Yield (ng)
37B	5.22	40	208.8
38B	1.29	40	51.6
40B	Out of range	40	0
41B	Out of range	40	0
42B	1.73	40	69.2
43B	0.46	40	18.4

RNA Quantification

20250325-gmac-qubit-RNA.hs-blood (Google Sheet)

Sample	Concentration (ng/uL)	Volume (uL)	Yield (ng)
37B	9.76	40	390.4
38B	Out of range	40	0
40B	Out of range	40	0
41B	Out of range	40	0
42B	17.5	40	700
43B	6.82	40	272.8

SUMMARY

Overall, I’d say yields were poor for both DNA and RNA. Samples 40B and 41B failed to yield any DNA and RNA.

For those DNA samples which yielded DNA, the yields are likely too low for any type of sequencing.

For those RNA samples which yielded RNA, the quantities are probably usable for sequencing; specifically the low input kits.

And, as a reminder, yields may have been impacted by my failure to add Proteinase K to the samples..