RNA Qubit tests

Concentrations for ITS2

We aim to do ITS2 for my ambient Mcap developmental timeseries samples from 2023 and for the POC spawning experimental samples from 2023. Mcap 2023 github repo is here and POC github is here.

When I did the original extractions, most of the samples did not have much DNA, according to Qubit DNA Broad Range. I’ve found that early life stages (ie embryos and larvae) typically don’t yield a lot of DNA, so this was not unexpected. Since I need to know the DNA concentrations for ITS2, I also nanodropped and Qubited DNA High Sensitivity the samples for the concentration. Both of these methods should provide concentrations, even if DNA concentration is low.

Below is a table with the Qubit Broad Range, Qubit High Sensitivity and Nanodrop concentrations for each sample.

TubeID hpf Treatment Project Qubit BR DNA1 (ng/uL) Qubit BR DNA2 (ng/uL) Qubit BR DNA Average (ng/uL) Qubit HS DNA1 (ng/uL) Qubit HS DNA2 (ng/uL) Qubit HS DNA Average (ng/uL) Nanodrop (ng/uL) Nanodrop notes Extraction post
M6 1 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.372 0.294 0.333 2.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M7 1 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.132 0.126 0.129 4.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M8 1 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.174 0.136 0.155 4.3   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M9 1 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.104 NA 0.104 0.3 Read multiple times, got mostly negative values (-1.3, -2.6, -1.3, -0.8, -1, -0.3, 0.6) https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M13 4 hpf Ambient Developmental Timeseries 2023 NA NA NA 1.05 0.928 0.989 4.1   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M10 4 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.132 0.132 0.132 7.8   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M11 4 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.14 0.132 0.136 3.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M14 4 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.288 0.238 0.263 6.4   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M23 9 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.116 NA 0.116 0.5 Also had a negative reading of -0.6 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M24 9 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.122 0.106 0.114 0.2 Also had a negative reading of -1.1 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M26 9 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.11 NA 0.11 1.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M28 9 hpf Ambient Developmental Timeseries 2023 NA NA NA NA NA NA 3.8   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M35 14 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.29 0.26 0.275 2.5   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M36 14 hpf Ambient Developmental Timeseries 2023 2.38 2.32 2.35 1.14 1.03 1.085 16   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M37 14 hpf Ambient Developmental Timeseries 2023 2 NA 2 1.05 0.904 0.977 7   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M39 14 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.452 0.468 0.46 3.7   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M52 22 hpf Ambient Developmental Timeseries 2023 NA NA NA 0.41 0.414 0.412 1 Also had readings of 31.6 and 59.8, these seemed very high https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M51 22 hpf Ambient Developmental Timeseries 2023 5.5 5.46 5.48 3.54 3.52 3.53 7.4   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M47 22 hpf Ambient Developmental Timeseries 2023 5.7 5.52 5.61 3 1.44 2.22 6.9   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M48 22 hpf Ambient Developmental Timeseries 2023 3.86 3.84 3.85 0.888 1.01 0.949 3.4   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M60 28 hpf Ambient Developmental Timeseries 2023 3.54 3.58 3.56 0.334 0.25 0.292 1.9 Also had reading of 0.3 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M62 28 hpf Ambient Developmental Timeseries 2023 6.56 6.52 6.54 9.74 9.3 9.52 4.5   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M63 28 hpf Ambient Developmental Timeseries 2023 5 4.92 4.96 4.58 4.36 4.47 8.2   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M61 28 hpf Ambient Developmental Timeseries 2023 10.6 10.5 10.55 8.64 8.68 8.66 13.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M72 48 hpf Ambient Developmental Timeseries 2023 4.58 4.66 4.62 0.872 0.824 0.848 0.4 Also had readings of 0.7 and -0.3 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-10-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M73 48 hpf Ambient Developmental Timeseries 2023 11 10.9 10.95 8.66 8.58 8.62 14.9   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M74 48 hpf Ambient Developmental Timeseries 2023 6.48 6.46 6.47 6.3 6.26 6.28 12.9   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M75 48 hpf Ambient Developmental Timeseries 2023 6.36 6.3 6.33 7.88 7.76 7.82 7.5   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M83 72 hpf Ambient Developmental Timeseries 2023 10.8 10.8 10.8 19.6 19.3 19.45 39.2   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-02-22-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M86 72 hpf Ambient Developmental Timeseries 2023 44.8 44.4 44.6 41.2 41 41.1 100.7 Also had reading of 104.4 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M87 72 hpf Ambient Developmental Timeseries 2023 28.6 28.4 28.5 29.8 29.6 29.7 38.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
M88 72 hpf Ambient Developmental Timeseries 2023 38.8 38.2 38.5 67.2 67.2 67.2 42.8   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-05-02-MiniprepPlus-DNA%3ARNA-extractions-McapLarvae-DT.md
6 Embryos Ambient POC NA NA NA 0.136 0.126 0.131 1.3 Also had negative readings of -0.2, -1.1, -0.4 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-03-27-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
7 Embryos Ambient POC NA NA NA 0.372 0.282 0.327 0.7 Also had readings of -0.3, -7.5, 0.5 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-03-27-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
8 Embryos Ambient POC NA NA NA 0.472 0.406 0.439 0.6 Also had readings of 0, -0.3, -0.5 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
9 Embryos Ambient POC NA NA NA 0.28 0.26 0.27 2.1   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
106 Larvae Ambient POC 3.48 3.46 3.47 1.01 0.566 0.788 2.7   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
107 Larvae High POC 2.34 2.24 2.29 0.59 0.574 0.582 Negative? Had all negative readings (-1, -0.5, -1.7) https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
108 Larvae Ambient POC NA NA NA 0.558 0.54 0.549 4 Also had reading of 0 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-03-27-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
109 Larvae High POC 4.54 4.56 4.55 2.88 2.66 2.77 4.7   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
110 Larvae Ambient POC 4.02 3.92 3.97 1.01 0.878 0.944 4   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-03-27-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
111 Larvae High POC 3.96 4 3.98 1.85 1.77 1.81 3.6   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
112 Larvae Ambient POC 3.52 3.52 3.52 1.73 1.69 1.71 3.8   https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-04-03-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md
113 Larvae High POC 2.8 2.7 2.75 1.31 1.18 1.245 1.2 Also had readings of -1 and 4.2 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-03-27-MiniprepPlus-DNA%3ARNA-extractions-Poc2023.md

Really all over the place…I trust the Qubit HS kit the most, given its ability to detect very low concentrations of DNA (ie 0.1 ng/uL). Nanodrop seemed unreliable and I got negative values for some samples, which is weird. I also read some samples multiple times and got very different values. The Qubit BR vs HS are also pretty different, which is interesting.

Next steps

Using the Qubit HS kit, I got DNA concentrations for all samples (except M28). My lowest concentration is M9 at 0.104 ng/uL. For ITS2, do I now need to dilute all samples to 0.104 ng/uL? Is 0.104 ng/uL enough material for ITS2? Will discuss w/ Hollie.

If I use my lowest sample: DNA final concentration = 0.104 ng/uL at volume of 10 uL = 1.04 ng.

TubeID hpf Treatment Project Qubit HS (ng/uL) DNA for dilution (uL) Water (uL) Total volume Final concentration (ng/uL) Notes
M6 1 hpf Ambient Developmental Timeseries 2023 0.333 3.12 6.88 10.00 0.104  
M7 1 hpf Ambient Developmental Timeseries 2023 0.129 8.06 1.94 10.00 0.104  
M8 1 hpf Ambient Developmental Timeseries 2023 0.155 6.71 3.29 10.00 0.104  
M9 1 hpf Ambient Developmental Timeseries 2023 0.104 10.00 0.00 10.00 0.104  
M13 4 hpf Ambient Developmental Timeseries 2023 0.989 1.05 8.95 10.00 0.104  
M10 4 hpf Ambient Developmental Timeseries 2023 0.132 7.88 2.12 10.00 0.104  
M11 4 hpf Ambient Developmental Timeseries 2023 0.136 7.65 2.35 10.00 0.104  
M14 4 hpf Ambient Developmental Timeseries 2023 0.263 3.95 6.05 10.00 0.104  
M23 9 hpf Ambient Developmental Timeseries 2023 0.116 8.97 1.03 10.00 0.104  
M24 9 hpf Ambient Developmental Timeseries 2023 0.114 9.12 0.88 10.00 0.104  
M26 9 hpf Ambient Developmental Timeseries 2023 0.11 9.45 0.55 10.00 0.104  
M28 9 hpf Ambient Developmental Timeseries 2023 NA NA NA NA NA May use 10 ng/uL DNA as input
M35 14 hpf Ambient Developmental Timeseries 2023 0.275 3.78 6.22 10.00 0.104  
M36 14 hpf Ambient Developmental Timeseries 2023 1.085 0.96 9.04 10.00 0.104  
M37 14 hpf Ambient Developmental Timeseries 2023 0.977 1.06 8.94 10.00 0.104  
M39 14 hpf Ambient Developmental Timeseries 2023 0.46 2.26 7.74 10.00 0.104  
M52 22 hpf Ambient Developmental Timeseries 2023 0.412 2.52 7.48 10.00 0.104  
M51 22 hpf Ambient Developmental Timeseries 2023 3.53 0.29 9.71 10.00 0.104  
M47 22 hpf Ambient Developmental Timeseries 2023 2.22 0.47 9.53 10.00 0.104  
M48 22 hpf Ambient Developmental Timeseries 2023 0.949 1.10 8.90 10.00 0.104  
M60 28 hpf Ambient Developmental Timeseries 2023 0.292 3.56 6.44 10.00 0.104  
M62 28 hpf Ambient Developmental Timeseries 2023 9.52 0.11 9.89 10.00 0.104  
M63 28 hpf Ambient Developmental Timeseries 2023 4.47 0.23 9.77 10.00 0.104  
M61 28 hpf Ambient Developmental Timeseries 2023 8.66 0.12 9.88 10.00 0.104  
M72 48 hpf Ambient Developmental Timeseries 2023 0.848 1.23 8.77 10.00 0.104  
M73 48 hpf Ambient Developmental Timeseries 2023 8.62 0.12 9.88 10.00 0.104  
M74 48 hpf Ambient Developmental Timeseries 2023 6.28 0.17 9.83 10.00 0.104  
M75 48 hpf Ambient Developmental Timeseries 2023 7.82 0.13 9.87 10.00 0.104  
M83 72 hpf Ambient Developmental Timeseries 2023 19.45 0.05 9.95 10.00 0.104  
M86 72 hpf Ambient Developmental Timeseries 2023 41.1 0.03 9.97 10.00 0.104  
M87 72 hpf Ambient Developmental Timeseries 2023 29.7 0.04 9.96 10.00 0.104  
M88 72 hpf Ambient Developmental Timeseries 2023 67.2 0.02 9.98 10.00 0.104  
6 Embryos Ambient POC 0.131 7.94 2.06 10.00 0.104  
7 Embryos Ambient POC 0.327 3.18 6.82 10.00 0.104  
8 Embryos Ambient POC 0.439 2.37 7.63 10.00 0.104  
9 Embryos Ambient POC 0.27 3.85 6.15 10.00 0.104  
106 Larvae Ambient POC 0.788 1.32 8.68 10.00 0.104  
107 Larvae High POC 0.582 1.79 8.21 10.00 0.104  
108 Larvae Ambient POC 0.549 1.89 8.11 10.00 0.104  
109 Larvae High POC 2.77 0.38 9.62 10.00 0.104  
110 Larvae Ambient POC 0.944 1.10 8.90 10.00 0.104  
111 Larvae High POC 1.81 0.57 9.43 10.00 0.104  
112 Larvae Ambient POC 1.71 0.61 9.39 10.00 0.104  
113 Larvae High POC 1.245 0.84 9.16 10.00 0.104  

Some of the DNA inputs are very low (0.02 for M88, 0.03 for M86) and I’m not confident that that volume of liquid can be pipetted accurately. Tomorrow, I may do a trial run with a couple of samples to see if this is feasible.

Written on May 8, 2024