RNA extraction and qPCR for snap frozen Lifestage Carryover samples

[AHuffmyer]

RNA extractions and qPCR for Lifestage Carryover project for 60 snap frozen samples.

These can be done in the same way as the previous round for the same targets (ATP synthase, Viperin, HSP70, HSP90, Citrate Synthase, cGAS, DMNT1 normalized to GADPH).

In the last round of analyses, we were not able to get RNA from some samples, leading to low sample size. This task is to conduct the extractions and qPCR on snap frozen samples to supplement sample size.

The adult group had sufficient sample sizes (n=8 per group), so here we will only use samples for spat, seed, and juveniles.

Note that the spat and seed samples are of whole individuals and will need to be crushed/homogenized during extractions.

The sample list can be found here. Please refer to the "notes" column for clarification on sample/tube IDs and freezer location.

I have attached pictures of the two boxes that contain all samples. Please refer to the spreadsheet linked above for the tube ID's to use. The boxes also contain "adult" samples that will not be processed. Tubes in the boxes are arranged in numerical order.

@kubu4 - does this sound ok? Let me know if I can help with clarifying any questions. Are we good on supplies for processing these 60 samples?

Samples are in the two "LCO Phys" boxes in the image below in the middle freezer (top shelf).

[kubu4]

Are we good on supplies for processing these 60 samples

Nope. We'll need to order more reagents.

I'll get the necessary stuff ordered and will tackle once we start getting supplies in.

[AHuffmyer]

@kubu4 what is the status of supply orders for the remaining qPCR samples? Thank you!

[kubu4]

We have received all supplies.

This process might be getting "outsourced." Discussed this briefly with @sr320 , but nothing's been finalized yet.

[AHuffmyer]

Pending Sam processing (maybe in April/May)

[AHuffmyer]

@kubu4 the new undergrad hires could assist with this

[kubu4]

Beginning to tackle this, this week and next with assistance from undergrads.

[kubu4]

OMG. The results from these extractions are bad. 😢

Please see the table below.

The 20241230_rna_extractions.csv) has been updated with this info.

Extractions used the entirety of the sample in nearly all instances, so there isn't a way to redo these.

Let me know if you want me to proceed with reverse transcription of those samples which yielded RNA.

tube_ID	tag_tank	lifestage	conditioning_treatment	acute_treatment	rna-concentration(ng/uL)
1	left-juvenile	juvenile	treated	ambient	0
12	right-juvenile	juvenile	control	high	6.6
18	right-juvenile	juvenile	control	ambient	38.4
28	left-juvenile	juvenile	treated	high	0
34	left-juvenile	juvenile	treated	ambient	0
39	right-juvenile	juvenile	control	high	16.1
43	right-juvenile	juvenile	control	high	85.2
49	right-juvenile	juvenile	control	high	172
53	left-juvenile	juvenile	treated	high	200
57	right-juvenile	juvenile	control	ambient	0
61	left-juvenile	juvenile	treated	high	0
64	left-juvenile	juvenile	treated	ambient	0
65	right-juvenile	juvenile	control	ambient	0
71	right-juvenile	juvenile	control	high	77
73	left-juvenile	juvenile	treated	high	32.2
75	right-juvenile	juvenile	control	ambient	31.4
79	right-juvenile	juvenile	control	ambient	14.9
81	left-juvenile	juvenile	treated	high	112
83	left-juvenile	juvenile	treated	ambient	0
98	left-juvenile	juvenile	treated	ambient	0
14	left-seed	seed	treated	ambient	5
15	left-seed	seed	treated	high	55.4
19	left-seed	seed	treated	high	23.6
24	left-seed	seed	treated	high	20.4
29	right-seed	seed	control	ambient	4.6
40	right-seed	seed	control	ambient	8.64
42	right-seed	seed	control	high	0
48	left-seed	seed	treated	ambient	0
55	right-seed	seed	control	ambient	0
59	right-seed	seed	control	high	60
60	right-seed	seed	control	high	162
62	right-seed	seed	control	high	82.4
63	right-seed	seed	control	ambient	5.36
66	left-seed	seed	treated	ambient	27.2
69	right-seed	seed	control	ambient	15.9
72	left-seed	seed	treated	ambient	0
86	right-seed	seed	control	high	0
88	left-seed	seed	treated	high	0
89	left-seed	seed	treated	ambient	6.42
92	left-seed	seed	treated	high	0
6	left-spat	spat	treated	high	0
9	left-spat	spat	treated	ambient	0
11	right-spat	spat	control	ambient	0
25	left-spat	spat	treated	high	6.72
27	right-spat	spat	control	high	0
30	right-spat	spat	control	ambient	0
36	right-spat	spat	control	ambient	0
38	left-spat	spat	treated	ambient	0
46	left-spat	spat	treated	ambient	0
47	left-spat	spat	treated	ambient	0
50	left-spat	spat	treated	high	0
52	right-spat	spat	control	ambient	0
74	right-spat	spat	control	high	0
76	left-spat	spat	treated	high	0
77	right-spat	spat	control	ambient	0
78	left-spat	spat	treated	high	0
93	right-spat	spat	control	high	0
96	right-spat	spat	control	high	0
97	right-spat	spat	control	high	0
13*	left-spat	spat	treated	ambient	0

[AHuffmyer]

Thats a bummer - I'll look at number of samples and let you know which to proceed with for qPCR!

[AHuffmyer]

@kubu4 please proceed with the samples that did yield sufficient RNA. This is distributed across a few treatment groups and will boost sample size from our previous extractions. Thank you!

[AHuffmyer]

@kubu4 what is the status of this round of qPCR? Thank you.

[kubu4]

These have been performed. Files were originally pushed upon completion in June.

I did realize a couple of weeks ago that the target names were inconsistent with the previous runs, so I updated those to match and pushed those a couple of weeks ago.

Analysis code has not been re-run.

[AHuffmyer]

Thanks! To confirm, all data is uploaded, scripts just need to be re-run?

[kubu4]

Yep.