RNA Quantification - C.gigas Lifestage Carryover RNA Samples Using Qubit High Sensitivity RNA Assay

2025
Crassostrea gigas
Pacific oyster
RNA quantification
Qubit
Magallana gigas
Author

Sam White

Published

May 13, 2025

INTRO

I was asked to isolate RNA from a list of 60 samples (GitHub Issue) from a number of Crassostrea gigas (Pacific oyster) spat and seed that had been collected as part of the project-gigas-carryover (GitHub repo). Yesterday, I homogenized samples in TriZol (Notebook entry), as well as isolated RNA from most of them (Notebook entry). I isolated RNA from the remaining 12 samples earlier today (Notebook entry).

Next on deck - quantification!

MATERIALS & METHODS

RNA was quantified with the Roberts Lab Qubit 3.0 fluorometer (Invitrogen). Samples were quantified with the Qubit RNA High Sensitivity (HS) assay (Invitrogen). 1uL of sample was used for the assay(s).

All RNA samples were stored in the -80oC boxes from where the original tissues came from.

RESULTS

Raw Qubit Data

Qubit (Google Sheets):

This info has been entered into the canonical CSV for these samples:

RNA Yields

Table 1: RNA yields taking into account samples which needed to be diluted and re-measured.
sample_ID qubit_concentration (ng/uL) dilution_factor final_concentration volume (uL) total (ng)
1 0 1 0 40 0
6 0 1 0 40 0
9 0 1 0 40 0
11 0 1 0 40 0
12 6.6 1 6.6 40 264
13* 0 1 0 40 0
14 5 1 5 40 200
15 55.4 10 554 40 22160
18 38.4 10 384 40 15360
19 23.6 1 23.6 40 944
24 20.4 1 20.4 40 816
25 6.72 1 6.72 40 268.8
27 0 1 0 40 0
28 0 1 0 40 0
29 4.6 1 4.6 40 184
30 0 1 0 40 0
34 0 1 0 40 0
36 0 1 0 40 0
38 0 1 0 40 0
39 16.1 1 16.1 40 644
40 8.64 1 8.64 40 345.6
42 0 1 0 40 0
43 85.2 1 85.2 40 3408
46 0 1 0 40 0
47 0 1 0 40 0
48 0 1 0 40 0
49 172 1 172 40 6880
50 0 1 0 40 0
52 0 1 0 40 0
53 200 1 200 40 8000
55 0 1 0 40 0
57 0 1 0 40 0
59 60 1 60 40 2400
60 162 1 162 40 6480
61 0 1 0 40 0
62 82.4 1 82.4 40 3296
63 5.36 1 5.36 40 214.4
64 0 1 0 40 0
65 0 1 0 40 0
66 27.2 10 272 40 10880
69 15.9 1 15.9 40 636
71 77 1 77 40 3080
72 0 1 0 40 0
73 32.2 1 32.2 40 1288
74 0 1 0 40 0
75 31.4 10 314 40 12560
76 0 1 0 40 0
77 0 1 0 40 0
78 0 1 0 40 0
79 14.9 1 14.9 40 596
81 112 1 112 40 4480
83 0 1 0 40 0
86 0 1 0 40 0
88 0 1 0 40 0
89 6.42 1 6.42 40 256.8
92 0 1 0 40 0
93 0 1 0 40 0
96 0 1 0 40 0
97 0 1 0 40 0
98 0 1 0 40 0

Lifestage RNA Concentrations

Table 2: RNA concentrations sorted by lifestage
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

SUMMARY

Well, overall, these results are not good. A surprisingly large number of samples failed to yield any RNA. And, to make matters worse, when we look at the results by lifestage, we see only a single spat sample yielded any RNA!

I’m having a difficult time coming up with any explanations for the overall poor results. There was nothing during the RNA isolation process to suggest that there might be any issues with any of the samples (e.g. no clogged columns). Although, reviewingprevious isolations also did not work particularly well (Notebook entry), as well as others which performed perfectly (Notebook entry), I suspect I fell into the trap of not diluting the homogenized samples with sufficient TriZol afterwards - which is the primary difference between the 20240319 isolations and the 20240624 isolations (Notebook entry).