Samples

1C4F, 2C4F, 3C4F = 123C4
- 1 min Qiagin Tissue Disruption Tubes
1L14F, 2L14F, 3L14F = 123L14
- 1 min Qiagin Tissue Disruption Tubes
1M4F, 2M4F, 3M4F = 123M4
- 1 min Qiagin Tissue Disruption Tubes
1H14F, 2H14F, 3H14F = 123H14
- 1 min Qiagin Tissue Disruption Tubes

Summary

Note

Success! I pooled 3 samples together and this yielded enough RNA!

Note

I also tested 3 different lysing protocols, and all worked more or less the same. I skipped the Proteinase K digestion as per the ‘cells’ sample prep instruction in the kit manual. I think I can add this back in if I’m finding protein impurities in the eluted RNA.

Extraction Notes

date: 13-AUG-2024

kit: Zymo Quick-DNA/RNA Miniprep Plus Kit

Thaw samples to room temp
Vortex to mix
Transfer to Qiagin Tissue Disruption Tube
Homogenize for 1 minute
Centrifuge at 16,000xg for 1 minute to get rid of bubbles
- Checked for complete lysis… still saw entact cells so I homogenized again
Honogenized for another 1 minute
Split into two aliquots of 700uL & transfer to two new nuclease-free tubes
Twizzled 123L14, 123 M4, and 123H14 for extra mechanical lysing
Added 700uL of DNA/RNA Lysis Buffer to each tube ( a total of 1400uL volume in each tube )
Vortex to mix for 5 seconds
Check to see that embryos have dissolved and liquid is clear
- 123C4, clear
- 123L14, clear
- 123M4, cell debris
- 123H14, clear

—> Proceed to purification

Sequentially transfer 2800uL of each sample in aliquots of 700uL into yellow Spin-Away Filter in a Collection Tube and centrifuge for 30s at 16,000xg SAVE THE FLOW THROUGH FOR RNA PURIFICATION by tipping flow through into falcon tube
Doubled volume of 100% (200 proof) ethanol to the RNA flow through in a falcon tube and vortexed to mix. End volume of flow through + ethanol = 5mL
Sequentially transfer RNA flow-through + ethanol in volumes =>700uL into the green Zymo Spin IIICG Column in a Collection Tube
Saved 1st 1400uL for protein

Warning

This took 7 passes!
Performed DNase I Treatment on green RNA spin column
```
DB = 75*4
print(DB)
```
```
[1] 300
```
```
Dnase = 5*4 
print(Dnase)
```
```
[1] 20
```
- I didn’t invert to mix… I always lose some volume ‘beads’ in the lid when I do this. Instead I flicked the tube to mix it and kept all the volume down in the cnical end of the tube. This worked well and I had a full 80uL of treatment per spin column to work with.
Warmed Zymo DNase-RNase Free Water to 55C in heat block before elution, and slowly dripped water directly over filter.

eluted DNA volume: 30uL in Zymo DNase-RNase Free Water

eluted RNA volume: 30uL in Zymo DNase-RNase Free Water

Nanodrop

RNA

sample_id	ng/uL	A260	A280	260/280	260/230
123C4	43.3	1.083	0.611	1.77	1.27
123H14	7.8	0.195	0.060	3.25	-0.94
123M4	90.8	2.271	1.125	2.02	1.86
123L14	16.0	0.399	0.196	2.04	1.99

Warning

123L9 is showing contamination… There is no distinct peak at 260 (like is visible with the other samples).

Qubit

Using the Invitrogen Thermo Fischer RNA High Sensitivity Assay Kit.

Prepared working solution for 3 samples and two standards:

199*6

[1] 1194

Qubit RNA HS reagent in DMSO = 1uL * 5 = 6uL

Qubit RNA HS Buffer = 199uL * 5 = 1194uL

RNA

standard 1: 91.79

standard 2: calibration error (even after two attempts… I plan to rerun these after getting fresh assay reagents)

RunID: 2024-08-14_043136

sample_id	qubit_rna_1	qubit_rna_2
123C4	47.6 ng/uL	47.6 ng/uL
123L14	19.6 ng/uL	19.7 ng/uL
123M4	124 ng/uL	122 ng/uL
123H14	27.6 ng/uL	27.6 ng/uL

Storage Location

RNA samples are stored in -80C ‘Old Friedman’ Sanyo freezer on shelf 1 in coral-embryo-leachate RNA wax freezer boxes with green label tape:

DNA samples (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate DNA wax freezer box with yellow label tape:

Protein flow-through (not quantified) are stored in -20C in JPG Lab FSH 236 in shelf 2 in coral-embryo-leachate PROTEIN wax freezer box with blue label tape: