Laura’s Notebook: qPCR with Actin primer set, etc.

Preparing previously sequenced larval samples

I spoke with Katherine last week about combining QuantSeq data from the previous run and the upcoming run. She advised (and I agreed) that it’s optimal for me to re-run those samples. The kits have changed slightly, in addition to me not DNasing the samples previously, her prepping the libraries etc. I have RNA in the -80, so I’m going to catch them up to the other samples today by Turbo DNasing them, and including in the qPCR re-do. Samples are: 34, 35, 37, 39, 41, 44, 45, 47.

DNA contamination re-check with Actin primer set

Today we received a new set of Actin primers that Sam had previously used to check for gDNA contamination. I tested them on one of my larval DNA samples, and voila! My positive control amplified, and the curves appear as expected.

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Rather than re-doing ALL samples, I did a sub-set, as I’d like to avoid more thawing/freezing cycles. Oddly, the + control didn’t amplify again with this new primer set.

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I discussed the issue with Steven and Sam. Since I did in fact DNase all my samples, I shall move ahead with the RNA concenrating and quality checking.

Quantifying remaining RNA samples after DNasing

In addition to re-running qPCR on 42 samples, I also measured [RNA] for samples that I had not yet quantified due to running out of Qubit tubes. The samples that I did qPCR on were mostly those that also needed quantifying (to minimize freeze/thaw), however I also did some other samples to include a random assortment.

Next step: concentrate dilute samples with Zymo Cleaner/Concentrator

The QuantSeq prep starts with up to 500 ng RNA in 5uL (or less). Katherine advised that I should use no less than 200 or 300 ng. So, here is how much RNA I have in 5 uL:

Tissue Cohort Treatment (pCO2, Temp) RNA Tube # [RNA] after Dnase treatment (ng/uL) ng RNA in 0.5 uL Concentrate?
Ctenidia Dabob Bay High 291 158.0 790.0
Ctenidia Dabob Bay High 292 29.6 148.0 X
Ctenidia Dabob Bay High 293 39.6 198.0 X
Ctenidia Dabob Bay High 294 110.0 550.0
Ctenidia Dabob Bay High 295 34.8 174.0 X
Ctenidia Dabob Bay High 296 180.0 900.0
Ctenidia Dabob Bay High 298 182.0 910.0
Ctenidia Dabob Bay High 299 50.4 252.0 X
Ctenidia Dabob Bay Ambient 301 75.8 379.0
Ctenidia Dabob Bay Ambient 302 62.4 312.0
Ctenidia Dabob Bay Ambient 306 136.0 680.0
Ctenidia Dabob Bay Ambient 304 200.0 1,000.0
Ctenidia Dabob Bay Ambient 305 75.2 376.0
Ctenidia Dabob Bay Ambient 303 95.2 476.0
Ctenidia Dabob Bay Ambient 307 89.4 447.0
Ctenidia Dabob Bay Ambient 308 73.6 368.0
Ctenidia Dabob Bay Ambient 309 170.0 850.0
Ctenidia Oyster Bay High 311 158.0 790.0
Ctenidia Oyster Bay High 312 90.6 453.0
Ctenidia Oyster Bay High 313 72.4 362.0
Ctenidia Oyster Bay High 314 42.2 211.0 X
Ctenidia Oyster Bay High 315 148.0 740.0
Ctenidia Oyster Bay High 316 146.0 730.0
Ctenidia Oyster Bay High 317 158.0 790.0
Ctenidia Oyster Bay High 318 174.0 870.0
Ctenidia Oyster Bay High 319 77.6 388.0
Ctenidia Oyster Bay Ambient 321 148.0 740.0
Ctenidia Oyster Bay Ambient 322 44.6 223.0 X
Ctenidia Oyster Bay Ambient 323 102.0 510.0
Ctenidia Oyster Bay Ambient 324 172.0 860.0
Ctenidia Oyster Bay Ambient 325 180.0 900.0
Ctenidia Oyster Bay Ambient 326 130.0 650.0
Ctenidia Oyster Bay Ambient 327 85.2 426.0
Ctenidia Oyster Bay Ambient 328 156.0 780.0
Ctenidia Oyster Bay Ambient 329 162.0 810.0
Ctenidia Fidalgo Bay High 331 42.2 211.0 X
Ctenidia Fidalgo Bay High 332 65.8 329.0
Ctenidia Fidalgo Bay High 333 78.6 393.0
Ctenidia Fidalgo Bay High 334 64.8 324.0
Ctenidia Fidalgo Bay High 335 180.0 900.0
Ctenidia Fidalgo Bay High 336 94.8 474.0
Ctenidia Fidalgo Bay High 337 194.0 970.0
Ctenidia Fidalgo Bay High 338 81.6 408.0
Ctenidia Fidalgo Bay High 339 77.2 386.0
Ctenidia Fidalgo Bay Ambient 341 89.6 448.0
Ctenidia Fidalgo Bay Ambient 342 162.0 810.0
Ctenidia Fidalgo Bay Ambient 343 114.0 570.0
Ctenidia Fidalgo Bay Ambient 344 25.0 125.0 X
Ctenidia Fidalgo Bay Ambient 345 190.0 950.0
Ctenidia Fidalgo Bay Ambient 346 43.6 218.0 X
Ctenidia Fidalgo Bay Ambient 347 69.0 345.0
Ctenidia Fidalgo Bay Ambient 348 54.4 272.0
Ctenidia Fidalgo Bay Ambient 349 82.0 410.0
Larvae Dabob Bay 10 Ambient 401 93.4 467
Larvae Dabob Bay 10 Ambient 402 114.0 570
Larvae Dabob Bay 10 Ambient 403 136.0 680
Larvae Dabob Bay 10 Ambient 404 112.0 560
Larvae Dabob Bay 10 High 411 72.6 363
Larvae Dabob Bay 10 High 412 31.2 156 X
Larvae Dabob Bay 10 High 413 130.0 650
Larvae Dabob Bay 10 High 414 168.0 840
Larvae Dabob Bay 6 Ambient 421 57.6 288
Larvae Dabob Bay 6 High 431b 83.0 415
Larvae Dabob Bay 6 High 432 74.0 370
Larvae Dabob Bay 6 High 434 63.0 315
Larvae Fidalgo Bay 10 Ambient 441 16.2 81 X
Larvae Fidalgo Bay 10 Ambient 442b 69.8 349
Larvae Fidalgo Bay 10 Ambient 443 60.2 301
Larvae Fidalgo Bay 10 Ambient 444 70.6 353
Larvae Fidalgo Bay 10 Ambient 445 160.0 800
Larvae Fidalgo Bay 10 High 451 68.4 342
Larvae Fidalgo Bay 10 High 452b 97.2 486
Larvae Fidalgo Bay 10 High 453 196.0 980
Larvae Fidalgo Bay 6 Ambient 461b 84.0 420
Larvae Fidalgo Bay 6 Ambient 462b 106.0 530
Larvae Fidalgo Bay 6 High 471b 108.0 540
Larvae Fidalgo Bay 6 High 472b 97.0 485
Larvae Fidalgo Bay 6 High 473 124.0 620
Larvae Fidalgo Bay 6 High 474 77.2 386
Larvae Fidalgo Bay 6 High 475 27.4 137 X
Larvae Fidalgo Bay 6 High 476 39.2 196 X
Larvae Fidalgo Bay 6 High 477 164.0 820
Larvae Oyster Bay C1 10 Ambient 481 89.2 446
Larvae Oyster Bay C1 10 Ambient 482 22.2 111 X
Larvae Oyster Bay C1 10 Ambient 483 99.0 495
Larvae Oyster Bay C1 10 Ambient 484 58.4 292
Larvae Oyster Bay C1 10 Ambient 485 19.1 96 X
Larvae Oyster Bay C1 10 Ambient 486b 148.0 740
Larvae Oyster Bay C1 10 Ambient 487 118.0 590
Larvae Oyster Bay C1 10 Ambient 488 60.0 300
Larvae Oyster Bay C1 10 Ambient 489 68.0 340
Larvae Oyster Bay C1 10 Ambient 490 186.0 930
Larvae Oyster Bay C1 10 Ambient 491 58.4 292
Larvae Oyster Bay C1 10 Ambient 492 82.6 413
Larvae Oyster Bay C1 10 High 41 108.0 540
Larvae Oyster Bay C1 10 High 43 156.0 780
Larvae Oyster Bay C1 10 High 46 98.6 493
Larvae Oyster Bay C1 10 High 47 128.0 640
Larvae Oyster Bay C1 10 High 44 41.4 207 X
Larvae Oyster Bay C1 10 High 506 29.2 146 X
Larvae Oyster Bay C1 10 High 45 25.1 126 X
Larvae Oyster Bay C1 6 Ambient 35 94.4 472
Larvae Oyster Bay C1 6 Ambient 38 LOW #VALUE!
Larvae Oyster Bay C1 6 Ambient 513 142.0 710
Larvae Oyster Bay C1 6 Ambient 39 89.8 449
Larvae Oyster Bay C1 6 Ambient 37 74.6 373
Larvae Oyster Bay C1 6 Ambient 34 104.0 520
Larvae Oyster Bay C1 6 High 521 66.6 333
Larvae Oyster Bay C1 6 High 522 32.2 161 X
Larvae Oyster Bay C1 6 High 523 71.4 357
Larvae Oyster Bay C1 6 High 524 63.0 315
Larvae Oyster Bay C1 6 High 525 140.0 700
Larvae Oyster Bay C1 6 High 526 138.0 690
Larvae Oyster Bay C1 6 High 527 124.0 620
Larvae Oyster Bay C1 6 High 528 87.6 438
Larvae Oyster Bay C1 6 High 529 56.8 284
Larvae Oyster Bay C2 10 Ambient 531 95.4 477
Larvae Oyster Bay C2 10 Ambient 532 93.6 468
Larvae Oyster Bay C2 10 Ambient 533 6.5 33 X
Larvae Oyster Bay C2 10 High 541 44.4 222 X
Larvae Oyster Bay C2 10 High 542 32.8 164 X
Larvae Oyster Bay C2 10 High 543 162.0 810
Larvae Oyster Bay C2 6 Ambient 551 96.4 482
Larvae Oyster Bay C2 6 Ambient 552b 74.6 373
Larvae Oyster Bay C2 6 Ambient 553 186.0 930
Larvae Oyster Bay C2 6 Ambient 554 188.0 940
Larvae Oyster Bay C2 6 High 561 28.0 140 X
Larvae Oyster Bay C2 6 High 562 126.0 630
Larvae Oyster Bay C2 6 High 563 47.0 235 X
Larvae Oyster Bay C2 6 High 564 152.0 760
Larvae Oyster Bay C2 6 High 565 31.2 156 X
Control homog. Control RNA Control 571 LOW LOW
Control homog. Control RNA Control 572
Control RNAzol control RNA Control 574
Control Turbo Dnase control RNA Control 575

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