More RNA extracted from Day 9 - RNA in all

I extracted RNA from the same tubes that I worked with a lot the past couple of weeks on the bioanalyzer and Nanodrop and qubit because there ended up being not much left. RNA was in all samples. Details of extraction and next steps in post.

Samples:

Because I used so much of the eluted RNA from the samples that I tried to get DNA free (post), I re-did those samples.

FRP trtmnt_tank day infection_status maturity tube_number
6157 NA 9 0 M 169
6143 NA 9 0 M 10
6228 NA 9 0 I 29
6150 NA 9 0 M 119
6277 NA 9 0 I 177
6156 NA 9 0 I 24
6272 NA 9 0 I 111
6178 NA 9 0 M 124
6161 NA 9 0 M 53
6136 NA 9 0 M 91
6137 NA 9 1 I 81
6112 MA 9 0 I 28

RNA isolation

Used the same process to isolate as previous posts with the Zymo RNA Microprep kit. No obvious, glaring mistakes were made as far as I’m aware.

Results:

I acciddentally didn’t make enough of the Qubit RNA HS working solution for all 12 samples plus the two standards, so I ran 10 samples, and then the remaining two separately.

Google sheets
Raw qubit (10 samples)
Raw qubit (2 sampels)

test_date qubit_tube_conc_ng.ml original_sample_conc_ng.ul sample_vol_ul dilution_factor tube_number extraction_method ul_sample-used elution_vol_ul total-yield_ng
11/15/19 14:57 157 15.7 2 100 91 Zymo_microprep 35 15 204.1
11/15/19 14:57 312 31.2 2 100 53 Zymo_microprep 35 15 405.6
11/15/19 14:57 228 22.8 2 100 124 Zymo_microprep 35 15 296.4
11/15/19 14:57 399 39.9 2 100 111 Zymo_microprep 35 15 518.7
11/15/19 14:56 282 28.2 2 100 24 Zymo_microprep 35 15 366.6
11/15/19 14:56 630 63 2 100 177 Zymo_microprep 35 15 819
11/15/19 14:56 323 32.3 2 100 119 Zymo_microprep 35 15 419.9
11/15/19 14:56 331 33.1 2 100 29 Zymo_microprep 35 15 430.3
11/15/19 14:56 229 22.9 2 100 10 Zymo_microprep 35 15 297.7
11/15/19 14:56 281 28.1 2 100 169 Zymo_microprep 35 15 365.3
11/15/19 15:04 221 22.1 2 100 28 Zymo_microprep 35 15 287.3
11/15/19 15:04 510 51 2 100 81 Zymo_microprep 35 15 663

RNA in all!!

Next steps:

Come up with pooling plan for the next 6 libraries:

Infection status Day Temp trtmnt
Infected 9 NA
Uninfected 9 NA
Infected 12 cold
Uninfected 12 cold
Infected 12 warm
Uninfected 12 warm

Pool them next week, and hopefully by then we’ll have a quote from NWGC, so that we can submit them next week for library prep and seq.

Written on November 15, 2019