RNA and DNA Extractions for E5 ACR Deep Dive Project, January 23, 2023

Protocol Link

Extraction of 18 ACR samples from Moorea from the E5 Time Series (Time Points 1 & 4, sampled January and November 2020)

Samples

(Note, the order of ACR-237 TP1 and TP4 are backward in this photo, but they were extracted in the right order.)

Notes

• I located these flash frozen samples on January 18 and clipped them following this protocol into 2.0 screw cap tubes with 1000 uL of DNA/RNA shield and 0.25 mL of 0.5mm glass beads. These were kept on dry ice during clipping and immediately put into the -80 ºC freezer.

• On extraction day, I did the following steps (homogenization very similar to how it is detailed in the protocol:
  1. Bead beat for 1-1.5 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
  2. Briefly spin down and remove 400 uL of supernatant into a clean tube. Spin for 3 mins at 9,000 rcf.
  3. Put original samples and bead tubes back in -80 ºC freezer.
  4. Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.
• Other than the adjustments mentioned above, I followed protocol exactly.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

Samples ACR-228 TP1 and ACR-234 TP1 were pale and were bead-beat for an extra 30 seconds to remove any last tissue. They had less tissue in the tubes than some of the other samples.

DNA Standards: 188.29 (S1) & 20735.98 (S2)

colony_id	timepoint	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
ACR-225	TP1	93	94.2	93.6
ACR-225	TP4	115	116	115.5
ACR-228	TP1	40	40.2	40.1
ACR-228	TP4	9.26	9.62	9.44
ACR-229	TP1	194	195	194.5
ACR-229	TP4	96.6	98.8	97.7
ACR-234	TP1	44.4	45.2	44.8
ACR-234	TP4	60.2	61.6	60.9
ACR-237	TP1	67.8	69.2	68.5
ACR-237	TP4	150	155	152.5
ACR-244	TP1	47.2	48	47.6
ACR-244	TP4	93.8	96.4	95.1
ACR-256	TP1	85.6	87	86.3
ACR-256	TP4	98.6	99.6	99.1
ACR-265	TP1	38.2	38.6	38.4
ACR-265	TP4	109	107	108
ACR-267	TP1	82.4	84.6	83.5
ACR-267	TP4	67.2	67.4	67.3

RNA Standards: 412.03 (S1) & 8733.93 (S2)

colony_id	timepoint	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
ACR-225	TP1	27.8	28.4	28.1
ACR-225	TP4	35	35.4	35.2
ACR-228	TP1	28	27.4	27.7
ACR-228	TP4	46.6	47.6	47.1
ACR-229	TP1	48.8	49.8	49.3
ACR-229	TP4	75	74.4	74.7
ACR-234	TP1	33.6	33.4	33.5
ACR-234	TP4	52	52	52
ACR-237	TP1	41.6	41.6	41.6
ACR-237	TP4	31.8	31.6	31.7
ACR-244	TP1	26.6	27	26.8
ACR-244	TP4	26.8	26.4	26.6
ACR-256	TP1	69.2	69.6	69.4
ACR-256	TP4	32.8	34	33.4
ACR-265	TP1	71.2	71.6	71.4
ACR-265	TP4	34.4	34.6	34.5
ACR-267	TP1	24.8	25	24.9
ACR-267	TP4	21.6	21.4	21.5

DNA and RNA Quality Check: Gel

Using Kristina’s gel protocol at the bottom of this page.

RNA Quality Check: Tapestation

I ran a tapestation on three of these samples (ACR-225 TP1, ACR-244 TP1, ACR-256 TP1) on 01/25/2023 to confirm the degradation of the RNA. We also included three of Kristina’s samples from the same set (one ACR #35 that did not work (ACR-234 TP1) and two that did: “41” (ACR-225 TP1) and “45” (ACR-256 TP1)). Hers were extracted from the molecular clippings preserved in the field in DNA/RNA shield.

Order:

Ladder	Kristina’s RNA	Kristina’s RNA	Kristina’s RNA	Zoe’s RNA	Zoe’s RNA	Zoe’s RNA
Ladder	35 (ACR-234 TP1)	41 (ACR-225 TP1)	45 (ACR-256 TP1)	ACR-244 TP1	ACR-225 TP1	ACR-256 TP1

Full results can be found here

Next steps

While the concentration of all samples looks good, the DNA and RNA both appear to be highly degraded on the gel. I will run them on the tapestation to try to better understand what is going on. Data (including Kristina’s original extraction data) are here (email me for access or request access at the link).

After running on the tapestation, the RNA appear severely degraded. We will reassess if we can utilize other samples that may not have thawed in transit. We could also try adjusting the homogenization technique and try immediately bead beating in DNA/RNA shield after clipping. The samples that Kristina used have been completely used up.