DNA and RNA Isolations - Gadus macrocephalus Blood

INTRO

Continued some testing samples for the Pacific cod project. In this instance, Steven and Laura asked that I extract DNA and RNA from the same blood sample (GitHub Issue). The idea was to determine how feasible this would be, specifically in regards to yields. The list of samples processed is here:

84       
93       
111      
114      
129      
149      
154      

Note

Sample 155 could not be located, despite being in the list of blood samples provided by Laura (GitHub Issue).

MATERIALS & METHODS

DNA and RNA Isolations

I used the Quick-DNA/RNA Microprep Plus Kit (ZymoResearch), and followed the manufacturer’s protocol (PDF) for whole blood extractions.

Note

• Used 200uL of DNA/RNA Shield for lysis.
• Used 8uL of Proteniase K for lysis.
• RNA was DNase-treated.
• DNA and RNA were each eluted with 15uL of H₂O.

I did encounter some issues, though. First, the tube lid/neck for Sample 114 snapped during an initial quick spin to collect blood off the sides of the tube:

Figure 1: Snapped microfuge tube lid/neck.

Figure 2: Sample 114 snapped microfuge tube lid/neck

Another issue was coagulation/incomplete lysis of virtually all of the samples:

Figure 3: Blood coagulation and incomplete lysis.

In retrospect, I probably should have attempted to physically homogenize the coagulated blood, but I did not.

DNA and RNA Quantifications

DNA and RNA were quantified using the Roberts Lab Qubit 3.0.

DNA was quantified using the dsDNA Broad Range Assay (Invitrogen). 1uL of each sample was used.

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

RESULTS

• DNA Quantification (Google Sheet)

Sample_ID	Concentration(ng/uL)
84	12.5
93	3.42
111	18.3
114	58.8
129	0
149	20.8
154	7.76

• RNA Quantification (Google Sheet)

Sample_ID	Concentration(ng/uL)
84	18.6
93	28
111	22.8
114	20.6
129	7.9
149	22.2
154	21.8

DISCUSSION

DNA yields were highly variable. RNA yields were more consistent. Overall, yields are likely lower for both sets, due to incomplete lysis, as noted above. Future attempts should involve physical homogenization to improve DNA/RNA recovery. Additionally, neither DNA or RNA were checked for integrity via gel and/or Bioanalyzer. I may investigate this at a later date.

Samples were temporarily stored in the middle -80^oC freezer, in a tube rack:

Figure 4: DNA and RNA tubes in orange tube rack on top shelf of middle -80^oC freezer.