Oly RNA isolation - larvae
July 23, 2019
Homogenized and frozen larvae to prep for RNA isolation, and aliquoted larve in ethanol for imaging/measuring at a later date. These larvae were collected during spring 2017, captured on screens upon release from mother, concentrated into microcentrifuge tubes, and placed immediately into a -80 freezer to preserve. Larvae in the same tube are likely to be full siblings or half siblings; the number of larvae collected on that day / from that collection bucket is a good indication of whether more than one female released brood that day (we expect ~200k larvae/female).
First step is homogenization, which is particularly necessary due to larvae having shells that need to be broken up. As with the ctenidia samples, I used mortar+pestle and liquid nitrogen. I pre-portioned 1 mL of RNAzol into microcentrifuge tubes, and transferred up to 100 mg homogenized larvae into chilled RNAzol. NOTE: I tried to get as much tissue as possible up to 100 mg, since the larval shells likely comprise a significant percentage of the mass.
In total I have 83 larval samples. I isolated RNA from 14 of these samples in spring 2018 for the test QuantSeq round. A few resulted in too little RNA, 2 of which had sufficient larvae remaining for another attempt to isolate RNA. So, I homogenized a total of 70 samples into RNAzol in 10 batches (~4 days of work), with an additional 2 controls (just liquid nitrogen ground into mortar+pestle).
July 24, 2019
Did a first round of RNA isolation (n=12, batch #1) to test protocol. Followed RNAzol protocol like with the ctenidia samples, with two exceptions: 1) after addition of DEPC-treatetd water (0.2 mL), centrifuged at 16,000 rcf (not 12,000); 2) added 100 uL DEPC-treated water in last step to dissolve RNA. Samples in this 1st batch: ABCDEFG
Also of note: after precipitating the DNA/proteins, the supernatant retained a black/gray color. Then, after adding isopropanol, the RNA pellet was predominantly black in 10 of the 12 samples (the 2 samples with small, white pellets were X and Y). Then, upon dissolving RNA in the DEPC-treated water and vortexing for 5 minutes, there was a black substance that settled into the bottom of the tubes. A black substance occurred before, in 2018 when I did a preliminary round of RNA isolation from the same study’s Oly larvae (see notebook post); this didn’t seem to interfere with the successful QuantSeq run.
I quantified RNA concentration in this first batch, using 1 uL of the RNA solution, pulled from the surface of the solution to avoid the black substance. Measured RNA in all samples, at a concentration of between 30-90 ng/uL.
Image of a few tubes showing that RNA pellete included black substance:
Image of RNA dissolved in water, showing a black substance settled at the bottom of the tubes.
July 26, 2019
Big day of RNA isolation - did 4 batches of 12, but processed 2 batches simultaneously, offset by about 10 minutes. This was feasible because there is down time during reactions/centrifuging.
Batches #2 and #3 (run simultaneously offset), followed the same protocol as with batch #1 (above), with one exception: added 75 uL of DEPC-treated water in final step to dissolve RNA.
Batch #2 samples: 402, 412, 421, 431, 432, 442, 452, 461, 462, 482, 532, 542
Batch #3 samples: 403, 404, 472, 473, 483, 484, 491, 522, 533, 552, 562, 571
Batches #4 and #5 (run simultaneously, offset), followed same protocol as batches 2 & 3, with one exception: did not vortex homogenate prior to transferring 500 uL to new tubes with the 200 uL DEPC-treated water. The resulting RNA dissolved in water had significantly less black substance, likely due to this adjustment.
I quantified RNA isolated from all samples, shown in the below table, and the spreadsheet is saved in the repo. A handful of samples resulted in relatively poor yield (452, 461, 462, 472, 533, 552), and several samples had a substantial amount of black substance mixed into the RNA solution (404, 431, 432, 442, 443(ish), 461, 462, 472, 486(ish), 521(ish), 522(ish), 552). Some samples in these these two categories overlapped - 461, 462, 472, 552. I will process some of the remaining homogenate (250 uL) to try to get a cleaner batch of RNA.
| Date larvae collected | Cohort | Treatment | TISSUE SAMPLE # | HOMOG. TUBE # | VOL RNAzol (mL) | MASS TISSUE (mg) | DATE HOMOG. | HOMOG. BATCH | RNA ISOLATION DATE | RNA ISOLATION BATCH | Total RNA volume remaining, uL (RNA + H2O) | [RNA] ng/uL | Amount of RNA (ng) | Volume needed for 500 ng RNA | Notes |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 5/24/17 | Dabob Bay | 10 Ambient | 14-A | 401 | 1 | 100 | 19-Jul | 1 | 24-Jul | 1 | 99 | 52.0 | 5,148 | 9.62 | |
| 5/31/17 | Dabob Bay | 10 Ambient | 31-A | 402 | 1 | 10 | 20-Jul | 3 | 26-Jul | 2 | 74 | 140.0 | 10,360 | 3.57 | |
| 6/19/17 | Dabob Bay | 10 Ambient | 75-A | 403 | 1 | 40 | 22-Jul | 5 | 26-Jul | 3 | 74 | 148.0 | 10,952 | 3.38 | |
| 6/29/17 | Dabob Bay | 10 Ambient | 80-A | 404 | 1 | 110 | 22-Jul | 6 | 26-Jul | 3 | 74 | 95.2 | 7,045 | 5.25 | |
| 5/26/17 | Dabob Bay | 10 Low | 23-A | 411 | 1 | 10 | 19-Jul | 1 | 24-Jul | 1 | 99 | 57.2 | 5,663 | 8.74 | |
| 5/27/17 | Dabob Bay | 10 Low | 27-A | 412 | 1 | 10 | 20-Jul | 3 | 26-Jul | 2 | 74 | 60.8 | 4,499 | 8.22 | |
| 6/10/17 | Dabob Bay | 10 Low | 58-A | 413 | 1 | 80 | 22-Jul | 7 | 26-Jul | 4 | 74 | 91.4 | 6,764 | 5.47 | |
| 6/12/17 | Dabob Bay | 10 Low | 60-A | 414 | 1 | 20 | 23-Jul | 8 | 26-Jul | 4 | 74 | 136.0 | 10,064 | 3.68 | |
| 6/12/17 | Dabob Bay | 6 Ambient | 59-A | 421 | 1 | 10 | 20-Jul | 2 | 26-Jul | 2 | 74 | 43.0 | 3,182 | 11.63 | |
| 6/7/17 | Dabob Bay | 6 Low | 51-A | 431 | 1 | 20 | 20-Jul | 2 | 26-Jul | 2 | 74 | 43.4 | 3,212 | 11.52 | |
| 6/17/17 | Dabob Bay | 6 Low | 72-A | 432 | 1 | 50 | 22-Jul | 4 | 26-Jul | 2 | 74 | 47.6 | 3,522 | 10.50 | |
| 6/17/17 | Dabob Bay | 6 Low | 73-A | 433 | na | na | na | na | na | na | na | na | na | na | |
| 6/19/17 | Dabob Bay | 6 Low | 74-A | 434 | 1 | 60 | 23-Jul | 9 | 5 | 74 | 67.8 | 5,017 | 7.37 | ||
| 5/25/17 | Fidalgo Bay | 10 Ambient | 20-A | 441 | 1 | 70 | 19-Jul | 1 | 24-Jul | 1 | 99 | 46.0 | 4,554 | 10.87 | |
| 6/3/17 | Fidalgo Bay | 10 Ambient | 38-A | 442 | 1 | 80 | 20-Jul | 3 | 26-Jul | 2 | 74 | LOW | |||
| 6/7/17 | Fidalgo Bay | 10 Ambient | 53-A | 443 | 1 | 60 | 22-Jul | 6 | 26-Jul | 4 | 74 | 58.4 | 4,322 | 8.56 | |
| 6/14/17 | Fidalgo Bay | 10 Ambient | 63-A | 444 | 1 | 80 | 22-Jul | 7 | 26-Jul | 4 | 74 | 91.2 | 6,749 | 5.48 | |
| 6/15/17 | Fidalgo Bay | 10 Ambient | 65-A | 445 | 1 | 40 | 23-Jul | 8 | 26-Jul | 5 | 74 | 132.0 | 9,768 | 3.79 | |
| 5/24/17 | Fidalgo Bay | 10 Low | 16-A | 451 | 1 | 70 | 19-Jul | 1 | 24-Jul | 1 | 99 | 68.4 | 6,772 | 7.31 | |
| 5/24/17 | Fidalgo Bay | 10 Low | 18-A | 452 | 1 | 80 | 20-Jul | 3 | 26-Jul | 2 | 74 | 37.8 | 2,797 | 13.23 | poor yield b/c black substance w/ RNA? |
| 6/3/17 | Fidalgo Bay | 10 Low | 36-A | 453 | 1 | 80 | 23-Jul | 9 | 27-Jul | 6 | 74 | 85.2 | 6,305 | 5.87 | |
| 5/26/17 | Fidalgo Bay | 6 Ambient | 22-A | 461 | 1 | 100 | 20-Jul | 2 | 26-Jul | 2 | 74 | 31.0 | 2,294 | 16.13 | poor yield b/c black substance w/ RNA? |
| 5/29/17 | Fidalgo Bay | 6 Ambient | 29-A | 462 | 1 | 60 | 22-Jul | 4 | 26-Jul | 2 | 74 | 29.8 | 2,205 | 16.78 | poor yield b/c black substance w/ RNA? |
| 5/25/17 | Fidalgo Bay | 6 Low | 19-A | 471 | 1 | 100 | 20-Jul | 2 | 24-Jul | 1 | 99 | 33.8 | 3,346 | 14.79 | |
| 5/26/17 | Fidalgo Bay | 6 Low | 21-A | 472 | 1 | 70 | 22-Jul | 4 | 26-Jul | 3 | 74 | 8.3 | 613 | 60.39 | poor yield b/c black substance w/ RNA? |
| 6/5/17 | Fidalgo Bay | 6 Low | 46-A | 473 | 1 | 50 | 22-Jul | 5 | 26-Jul | 3 | 74 | 120.0 | 8,880 | 4.17 | |
| 6/5/17 | Fidalgo Bay | 6 Low | 47-A | 474 | 1 | 50 | 22-Jul | 6 | 26-Jul | 4 | 74 | 81.0 | 5,994 | 6.17 | |
| 6/6/17 | Fidalgo Bay | 6 Low | 50-A | 475 | 1 | 80 | 22-Jul | 7 | 26-Jul | 4 | 74 | 110.0 | 8,140 | 4.55 | |
| 6/10/17 | Fidalgo Bay | 6 Low | 54-A | 476 | 1 | 80 | 23-Jul | 8 | 26-Jul | 5 | 74 | 43.4 | 3,212 | 11.52 | |
| 6/19/17 | Fidalgo Bay | 6 Low | 76-A | 477 | 1 | 40 | 23-Jul | 9 | 27-Jul | 6 | 74 | HIGH | |||
| 5/20/17 | Oyster Bay C1 | 10 Ambient | 02-A, 02-B | 481 | 1 | 40 | 19-Jul | 1 | 24-Jul | 1 | 99 | 64.4 | 6,376 | 7.76 | |
| 5/20/17 | Oyster Bay C1 | 10 Ambient | 04-A, 04-B | 482 | 1 | 60 | 20-Jul | 3 | 26-Jul | 2 | 74 | 67.2 | 4,973 | 7.44 | |
| 5/21/17 | Oyster Bay C1 | 10 Ambient | 03-A, 03-B | 483 | 1 | 110 | 22-Jul | 4 | 26-Jul | 3 | 74 | 95.8 | 7,089 | 5.22 | |
| 5/23/17 | Oyster Bay C1 | 10 Ambient | 09-A | 484 | 1 | 40 | 22-Jul | 5 | 26-Jul | 3 | 74 | 66.2 | 4,899 | 7.55 | |
| 6/1/17 | Oyster Bay C1 | 10 Ambient | 34-A | 485 | 1 | 20 | 22-Jul | 6 | 26-Jul | 4 | 74 | 156.0 | 11,544 | 3.21 | |
| 6/3/17 | Oyster Bay C1 | 10 Ambient | 39-A | 486 | 1 | 90 | 22-Jul | 7 | 26-Jul | 4 | 74 | 41.0 | 3,034 | 12.20 | |
| 6/3/17 | Oyster Bay C1 | 10 Ambient | 40-A | 487 | 1 | 30 | 23-Jul | 8 | 26-Jul | 5 | 74 | 112.0 | 8,288 | 4.46 | |
| 6/4/17 | Oyster Bay C1 | 10 Ambient | 44-A | 488 | 1 | 70 | 23-Jul | 9 | 26-Jul | 5 | 74 | 57.2 | 4,233 | 8.74 | |
| 6/6/17 | Oyster Bay C1 | 10 Ambient | 49-A | 489 | 1 | 10 | 23-Jul | 9 | 27-Jul | 6 | 74 | 57.2 | 4,233 | 8.74 | |
| 6/14/17 | Oyster Bay C1 | 10 Ambient | 64-A | 490 | 1 | 70 | 22-Jul | 7 | 27-Jul | 6 | 74 | 58.6 | 4,336 | 8.53 | |
| 6/15/17 | Oyster Bay C1 | 10 Ambient | 66-A | 491 | 1 | 20 | 22-Jul | 5 | 26-Jul | 3 | 74 | 126.0 | 9,324 | 3.97 | |
| 7/6/17 | Oyster Bay C1 | 10 Ambient | 81-A | 492 | 1 | 70 | 22-Jul | 6 | 26-Jul | 5 | 74 | 122.0 | 9,028 | 4.10 | |
| 5/21/17 | Oyster Bay C1 | 10 Low | 06-A, 06-B | 501 | 1 | <10 | 19-Jul | 1 | na | na | na | na | na | na | |
| 5/23/17 | Oyster Bay C1 | 10 Low | 08-A | 502 | na | na | na | na | na | na | na | na | na | na | |
| 5/26/17 | Oyster Bay C1 | 10 Low | 24-A | 503 | na | na | na | na | na | na | na | na | na | na | |
| 5/27/17 | Oyster Bay C1 | 10 Low | 26-A | 504 | na | na | na | na | na | na | na | na | na | na | |
| 5/31/17 | Oyster Bay C1 | 10 Low | 32-A | 505 | na | na | na | na | na | na | na | na | na | na | |
| 6/14/17 | Oyster Bay C1 | 10 Low | 62-A | 506 | 1 | 80 | 20-Jul | 2 | 24-Jul | 1 | 99 | 63.8 | 6,316 | 7.84 | |
| 6/15/17 | Oyster Bay C1 | 10 Low | 67-A | 507 | na | na | na | na | na | na | na | na | na | na | |
| 6/24/17 | Oyster Bay C1 | 10 Low | 79-A | 508 | na | na | na | na | na | na | na | na | na | na | |
| 5/23/17 | Oyster Bay C1 | 6 Ambient | 10-A | 511 | na | na | na | na | na | na | na | na | na | na | |
| 6/3/17 | Oyster Bay C1 | 6 Ambient | 37-A | 512 | na | na | na | na | na | na | na | na | na | na | |
| 6/5/17 | Oyster Bay C1 | 6 Ambient | 45-A | 513 | 30 | 23-Jul | 10 | 26-Jul | 5 | 74 | 156.0 | 11,544 | 3.21 | ||
| 6/6/17 | Oyster Bay C1 | 6 Ambient | 48-A | 514 | na | na | na | na | na | na | na | na | na | na | |
| 6/15/17 | Oyster Bay C1 | 6 Ambient | 69-A | 515 | na | na | na | na | na | na | na | na | na | na | |
| 6/17/17 | Oyster Bay C1 | 6 Ambient | 71-A | 516 | na | na | na | na | na | na | na | na | na | na | |
| 6/19/17 | Oyster Bay C1 | 6 Ambient | 77-A | 517 | na | na | na | na | na | na | na | na | na | na | |
| 5/21/17 | Oyster Bay C1 | 6 Low | 01-A, 01-B- 01-C | 521 | 1 | 70 | 20-Jul | 2 | 24-Jul | 1 | 99 | 54.4 | 5,386 | 9.19 | |
| 5/22/17 | Oyster Bay C1 | 6 Low | 07-A | 522 | 1 | 20 | 22-Jul | 5 | 26-Jul | 3 | 74 | 60.8 | 4,499 | 8.22 | |
| 5/27/17 | Oyster Bay C1 | 6 Low | 25-A | 523 | 1 | 30 | 22-Jul | 6 | 26-Jul | 4 | 74 | 60.6 | 4,484 | 8.25 | |
| 5/27/17 | Oyster Bay C1 | 6 Low | 28-A | 524 | 1 | 80 | 22-Jul | 7 | 26-Jul | 4 | 74 | 80.8 | 5,979 | 6.19 | |
| 5/29/17 | Oyster Bay C1 | 6 Low | 30-A | 525 | 1 | 30 | 23-Jul | 8 | 26-Jul | 5 | 74 | 128.0 | 9,472 | 3.91 | |
| 5/31/17 | Oyster Bay C1 | 6 Low | 33-A | 526 | 1 | 30 | 23-Jul | 9 | 27-Jul | 6 | 74 | 65.2 | 4,825 | 7.67 | |
| 6/14/17 | Oyster Bay C1 | 6 Low | 61-A | 527 | 1 | 90 | 22-Jul | 6 | 27-Jul | 6 | 74 | 81.4 | 6,024 | 6.14 | |
| 6/15/17 | Oyster Bay C1 | 6 Low | 68-A | 528 | 1 | 30 | 23-Jul | 8 | 26-Jul | 5 | 74 | 162.0 | 11,988 | 3.09 | |
| 6/17/17 | Oyster Bay C1 | 6 Low | 70-A | 529 | 1 | 70 | 23-Jul | 9 | 26-Jul | 5 | 74 | 73.4 | 5,432 | 6.81 | |
| 5/24/17 | Oyster Bay C2 | 10 Ambient | 17-A | 531 | 1 | 60 | 19-Jul | 1 | 24-Jul | 1 | 99 | 88.2 | 8,732 | 5.67 | |
| 6/3/17 | Oyster Bay C2 | 10 Ambient | 42-A | 532 | 1 | 40 | 20-Jul | 3 | 26-Jul | 2 | 74 | 158.0 | 11,692 | 3.16 | |
| 6/10/17 | Oyster Bay C2 | 10 Ambient | 56-A | 533 | 1 | <10 | 22-Jul | 4 | 26-Jul | 3 | 74 | 7.4 | 548 | 67.57 | poor yield b/c very little tissue (likely) |
| 5/23/17 | Oyster Bay C2 | 10 Low | 12-A | 541 | 1 | 40 | 10-Jul | 1 | 24-Jul | 1 | 99 | 45.6 | 4,514 | 10.96 | |
| 5/24/17 | Oyster Bay C2 | 10 Low | 13-A | 542 | 1 | 30 | 20-Jul | 3 | 26-Jul | 2 | 74 | 82.0 | 6,068 | 6.10 | |
| 6/4/17 | Oyster Bay C2 | 10 Low | 43-A | 543 | 1 | 80 | 22-Jul | 7 | 27-Jul | 6 | 74 | 61.4 | 4,544 | 8.14 | |
| 6/1/17 | Oyster Bay C2 | 6 Ambient | 35-A | 551 | 1 | 30 | 20-Jul | 2 | 24-Jul | 1 | 99 | 86.0 | 8,514 | 5.81 | |
| 6/3/17 | Oyster Bay C2 | 6 Ambient | 41-A | 552 | 1 | 80 | 22-Jul | 5 | 26-Jul | 3 | 74 | 17.5 | 1,295 | 28.57 | poor yield b/c black substance w/ RNA? |
| 6/10/17 | Oyster Bay C2 | 6 Ambient | 55-A | 553 | 1 | 30 | 23-Jul | 8 | 26-Jul | 4 | 74 | 200.0 | 14,800 | 2.50 | |
| 6/20/17 | Oyster Bay C2 | 6 Ambient | 78-A | 554 | 1 | 30 | 23-Jul | 9 | 26-Jul | 5 | 74 | 156.0 | 11,544 | 3.21 | |
| 5/21/17 | Oyster Bay C2 | 6 Low | 05-A | 561 | 1 | 40 | 20-Jul | 2 | 24-Jul | 1 | 99 | 43.4 | 4,297 | 11.52 | |
| 5/23/17 | Oyster Bay C2 | 6 Low | 11-A | 562 | 1 | 90 | 22-Jul | 5 | 26-Jul | 3 | 74 | 106.0 | 7,844 | 4.72 | |
| 5/24/17 | Oyster Bay C2 | 6 Low | 15-A | 563 | 1 | 50 | 22-Jul | 6 | 26-Jul | 4 | 74 | 84.6 | 6,260 | 5.91 | |
| 6/7/17 | Oyster Bay C2 | 6 Low | 52-A | 564 | 1 | not recorded | 22-Jul | 7 | 27-Jul | 6 | 74 | HIGH | |||
| 6/10/17 | Oyster Bay C2 | 6 Low | 57-A | 565 | 1 | 10 | 23-Jul | 8 | 27-Jul | 6 | 74 | 31.8 | 2,353 | 15.72 | |
| NA | RNA Control | RNA Control | 571 | 1 | 10 | 22-Jul | 4 | 26-Jul | 3 | 74 | LOW | NA | NA | ||
| NA | RNA Control | RNA Control | 572 | 1 | 10 | 23-Jul | 10 | 26-Jul | 5 | 74 | LOW | NA | NA | ||
| NA | RNA Control | RNA Control | 574 | NA | NA | NA | NA | 27-Jul | 6 | 74 | LOW | NA | NA |