Montipora Larvae DNA/RNA Extraction 2

Using Larvae Extraction Protocol on 10 of Ariana’s Montipora Coral Larvae Stored in DNA/RNA Shield

Goal: Use optimized extraction method on 10 samples for Ariana’s project
Results: Good DNA and RNA quantity and quality, variable RNA purity
Take aways: Scaling up worked well, I am worried about the RNA purity in the last few samples. But the method was exactly the same for all of them, so I am not sure why it is bad in some samples. Maybe 3 will need to be extracted again because of the poor RNA quality, and maybe 12 will need to be extracted again to try to get DNA.

Sample Info:

Sample Timepoint TP Volume Shield in Storage # Larvae
D3 1hpf TP1 1mL 250ul eggs
D10 38hpf TP3 1mL note the spreadsheet says 500ul 50ul
D12 65hpf TP4 1mL note the spreadsheet says 500ul 50ul
D16 65hpf TP4 500ul 50ul
D18 93hpf TP5 500ul 50ul
D20 93hpf TP5 500ul 50ul
D26 183hpf TP7 300ul 20-30 individuals
D28 183hpf TP7 300ul 20-30 individuals
D32 183hpf TP9 300ul 15-20 individuals
D39 231hpf TP10 300ul 15-20 individuals

Based off of the sample info and how many larvae were present in each tube, save fractions were made for samples D3, D10, D12, D16, D18, and D20. No fractions were saved for D26, D28, D32, and D39.

Sample Volume Shield Added at Extraction Save Fraction? Saved Volume
D3 0 yes 500ul
D10 0 yes 500ul
D12 0 yes 500ul
D16 500ul yes 500ul
D18 500ul yes 500ul
D20 500ul yes 500ul
D26 200ul no -
D28 200ul no -
D32 200ul no -
D39 200ul no -

Extraction

  • Extraction protocol followed exactly
  • Extraction proceeded with 500ul of each sample
  • 500ul of DNA/RNA lysis buffer was used for each sample
  • 1mL of 100% ethanol was used for each sample

QC 20210318

Qubit

  • Broad Range dsDNA and High Sensitivity RNA Qubit protocol
  • All samples were read twice
  • One RNA sample needed to be read with the broad range, and I read D10 again as well because it was at the upper limit of the HS kit

BR DNA:

Sample Standard 1 Standard 2 DNA 1 ng/ul DNA 2 ng/ul Average ng/ul
D3 173 19382 too low - -
D10 - - 3.46 3.6 3.5
D12 - - too low - -
D16 - - 24.4 24.4 24.4
D18 - - 24.2 24.2 24.2
D20 - - 33.8 33.4 33.6
D26 - - 39.6 39.8 39.7
D28 - - 20.8 21.2 21
D32 - - 5.88 6.02 5.95
D39 - - 6.12 5.86 5.99

HS RNA:

Sample Standard 1 Standard 2 RNA 1 ng/ul RNA 2 ng/ul Average ng/ul
D3 46.49 785 too high - -
D10 - - 104 104 104
D12 - - 84 84.4 84.2
D16 - - 96.2 95.8 96
D18 - - 55.2 55 55.1
D20 - - 68.2 68 68.1
D26 - - 45.4 45.4 45.4
D28 - - 28.8 29.6 29.2
D32 - - 14.3 14.4 14.35
D39 - - 13.2 12.8 13

BR RNA:

Sample Standard 1 Standard 2 RNA 1 ng/ul RNA 2 ng/ul Average ng/ul
D3 387 8079 562 560 561
D10 - - 126 126 126

NanoDrop

Sample 260/230 260/280
D3 2.23 2.13
D10 1.80 2.09
D12 1.69 2.05
D16 1.64 2.07
D18 1.49 2.01
D20 1.68 2.06
D26 0.37 2.06
D28 1.33 1.96
D32 0.89 1.77
D39 0.86 1.69

Full Results:

Traces:

Gel

TapeStation

Sample RIN score
D3 5.6
D10 8.6
D12 8.6
D16 8.9
D18 8.9
D20 9.0
D26 9.2
D28 9.0
D32 9.0
D39 9.0
Written on March 17, 2021