Using Larvae Extraction Protocol on 5 of Ariana’s Montipora Coral Recruits on Flash Frozen Plugs

Goal: Use optimized extraction method on 6 plug samples and share 1 72hr plug with Kevin (doing metabolomics extractions), I did not do any bead homogenization this time
Results: Good DNA and RNA quantity, RNA quality is ok, and DNA quality is less smeared than last time
Take aways: RNA quality and purity was ok, DNA is still smeared but not as bad as with the bead beating. The scraping from the plugs must be very harsh for it or something.

Sample Info

Note, the whirl packs didn’t have numbers so I gave them IDs, some plugs had a number on them

Sample	Timepoint	Number
Plug_4	48hps	98
Plug_5	48hps	57
Plug_6	72hps	–
Plug_7	48hps	–
Plug_8	5dps	461

Notes

• I did not save any of the plug, I used all of it because there was little material on each one
• I followed the same scrapping protocol as the previous post except that I did not do the bead homogenization, only vortexing
• The RNA was eluted in 70ul ultrapure water

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
• All samples were eluted for RNA with 40ul ultrapure water in the first elution and 30ul in the second

QC

Qubit

• Broad Range dsDNA and High Sensitivity RNA Qubit protocol
• All samples were read twice

BR DNA:

Sample	Standard 1	Standard 2	DNA 1 ng/ul	DNA 2 ng/ul	Average ng/ul
Plug_4	177	19116	1.2	50.8	51
Plug_5	-	-	19.9	19.6	19.75
Plug_6	-	-	44.2	43.4	42.8
Plug_7	-	-	30.2	29.6	29.9
Plug_8	-	-	37.8	37.4	37.6

HS RNA:

Sample	Standard 1	Standard 2	RNA 1 ng/ul	RNA 2 ng/ul	Average ng/ul
Plug_4	45	765	80.6	79.6	80.1
Plug_5	-	-	77.4	77.6	77.5
Plug_6	-	-	27.2	27.2	27.2
Plug_7	-	-	53.2	52.8	53
Plug_8	-	-	56.4	56.2	56.3

NanoDrop

• Followed NanoDrop RNA Protocol

Sample	260/230	260/280
Plug_4	1.39	1.94
Plug_5	1.01	2.03
Plug_6	1.01	1.79
Plug_7	1.14	1.88
Plug_8	1.28	1.96

Gel

• 1% gel run for 1 hour at 100V
• Gel Protocol

TapeStation

• Followed RNA TapeStation protocol

Sample	RIN score
Plug_4	7.4
Plug_5	7
Plug_6	6.9
Plug_7	6.7
Plug_8	7.4

Results Link