QuantSeq Purification and qPCR Assay Batches 3, 4, 5 (larvae)

I purified the ds cDNA from batches 3, 4 and 5. I then ran the qPCR assay on all samples to identify the optimal number of cycles.

Here is the plate configuration (showing sample numbers) for both the purified cDNA libraries, and for the qPCR assay

Column1 1 2 3 4 5 6 7 8 9 10 11 12
A 35 533 562 527 571 490 421 43 451 403 471b 401
B 524 522 432 492 525 523 553 506 572 461b 39 513
C 453 474 37 475 563 473 41 482 472b 431b 521 441
D 554 452b 413 541 404 485 46 412 414 564 411 444
E 442b 443 45 565 484 402 551 488 528 532 529 44
F 489 477 561 B3 - NTC1 531 487 486b 47 483 481 552b B5 - NTC1
G 462b 526 542 B3 - NTC2 34 476 445 B4 - NTC2 543 491 434 B5 - NTC2
H                        

image

End point PCR cycle calcuations

Batch 3

Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used RFU @ endpoint 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
35 94.4 3.71 1.29 350 3337 1,669 22.76 19.76 19
524 63.0 5.00 - 315 3144 1,572 17.51 14.51 14
453 196.0 1.79 3.21 350 3270 1,635 17.38 14.38 14
554 188.0 1.86 3.14 350 3198 1,599 17.6 14.6 14
442b 69.8 5.00 - 349 3162 1,581 20.39 17.39 17
489 68.0 5.00 - 340 3220 1,610 18.15 15.15 15
462b 106.0 3.30 1.70 350 3612 1,806 19.1 16.1 16
533 6.5 5.00 - 32.6 2713 1,357 20.11 17.11 17
522 32.2 5.00 - 161 2469 1,235 19.11 16.11 16
474 77.2 4.53 0.47 350 3547 1,774 17.58 14.58 14
452b 97.2 3.60 1.40 350 2770 1,385 19.07 16.07 16
443 60.2 5.00 - 301 3799 1,900 18.58 15.58 15
477 164.0 2.13 2.87 350 2498 1,249 18 15 15
526 138.0 2.54 2.46 350 3023 1,512 17.68 14.68 14
562 126.0 2.78 2.22 350 3063 1,532 20.09 17.09 17
432 74.0 4.73 0.27 350 2742 1,371 20.59 17.59 17
37 74.6 4.69 0.31 350 3451 1,726 23.6 20.6 20
413 130.0 2.69 2.31 350 2715 1,358 18.07 15.07 15
45 25.1 5.00 - 125.5 3349 1,675 23.73 20.73 20
561 28.0 5.00 - 140 2520 1,260 19.95 16.95 17
542 32.8 5.00 - 164 2999 1,500 19.66 16.66 16
527 124.0 2.82 2.18 350 3100 1,550 18.1 15.1 15
492 82.6 4.24 0.76 350 2747 1,374 18.42 15.42 15
475 27.4 5.00 - 137 1939 970 17.65 14.65 14
541 44.4 5.00 - 222 2387 1,194 18.76 15.76 15
565 31.2 5.00 - 156 2907 1,454 20.11 17.11 17
B3 - NTC1 NA - 5.00 #VALUE! 2959 1,480 26.88 23.88 23
B3 - NTC2 NA - 5.00 #VALUE! 2933 1,467 26.73 23.73 23

Batch 4

Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used RFU @ endpoint 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
571 LOW 5.00 - NA 3035 1,518 21.65 18.65 18
525 140.0 2.50 2.50 350 3283 1,642 17.05 14.05 14
563 47.0 5.00 - 235 3233 1,617 18.41 15.41 15
404 112.0 3.13 1.88 350 2712 1,356 18.16 15.16 15
484 58.4 5.00 - 292 2968 1,484 18.59 15.59 15
531 95.4 3.67 1.33 350 2434 1,217 18.24 15.24 15
34 104.0 3.37 1.63 350 2831 1,416 21.25 18.25 18
490 186.0 1.88 3.12 350 2913 1,457 17.12 14.12 14
523 71.4 4.90 0.10 350 2733 1,367 19.79 16.79 16
473 124.0 2.82 2.18 350 3055 1,528 17.63 14.63 14
485 19.1 5.00 - 95.5 3053 1,527 17.08 14.08 14
402 114.0 3.07 1.93 350 3246 1,623 19.16 16.16 16
487 118.0 2.97 2.03 350 2730 1,365 19.84 16.84 16
476 39.2 5.00 - 196 2546 1,273 16.7 13.7 13
421 57.6 5.00 - 288 2504 1,252 17.3 14.3 14
553 186.0 1.88 3.12 350 2588 1,294 17.23 14.23 14
41 108.0 3.24 1.76 350 2820 1,410 20.61 17.61 17
46 98.6 3.55 1.45 350 2998 1,499 21.83 18.83 18
551 96.4 3.63 1.37 350 3222 1,611 17.45 14.45 14
486b 148.0 2.36 2.64 350 3150 1,575 19.35 16.35 16
445 160.0 2.19 2.81 350 3349 1,675 18.27 15.27 15
43 156.0 2.24 2.76 350 3271 1,636 17.92 14.92 15
506 29.2 5.00 - 146 2556 1,278 18.51 15.51 15
482 22.2 5.00 - 111 2830 1,415 17.88 14.88 14
412 31.2 5.00 - 156 3599 1,800 20.56 17.56 17
488 60.0 5.00 - 300 3447 1,724 17.85 14.85 14
47 128.0 2.73 2.27 350 2863 1,432 20.01 17.01 17
B4 - NTC2 NA - 5.00 0 3554 1,777 25.29 22.29 22

Batch 5

Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used RFU @ endpoint 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
451 68.4 5.12 (0.12) 350 2895 1,448 17.73 14.73 14
572 LOW - 5.00 NA 2734 1,367 26.54 23.54 23
472b 97.0 3.61 1.39 350 2718 1,359 18.06 15.06 15
414 168.0 2.08 2.92 350 2951 1,476 18.42 15.42 15
528 87.6 4.00 1.00 350 3299 1,650 16.23 13.23 13
483 99.0 3.54 1.46 350 2845 1,423 19.19 16.19 16
543 162.0 2.16 2.84 350 3984 1,992 17.22 14.22 14
403 136.0 2.57 2.43 350 3065 1,533 17.5 14.5 14
461b 84.0 4.17 0.83 350 2122 1,061 17.57 14.57 14
431b 83.0 4.22 0.78 350 3181 1,591 19.24 16.24 16
564 152.0 2.30 2.70 350 3101 1,551 17.55 14.55 14
532 93.6 3.74 1.26 350 3494 1,747 17.47 14.47 14
481 89.2 3.92 1.08 350 1949 975 17.76 14.76 14
491 58.4 5.00 - 292 3545 1,773 18.23 15.23 15
471b 108.0 3.24 1.76 350 2470 1,235 18.52 15.52 15
39 89.8 3.90 1.10 350 2458 1,229 18.27 15.27 15
521 66.6 5.00 - 333 4101 2,051 20.44 17.44 17
411 72.6 4.82 0.18 350 3563 1,782 19.5 16.5 16
529 56.8 5.00 - 284 4097 2,049 18.65 15.65 15
552b 74.6 4.69 0.31 350 2970 1,485 18.79 15.79 15
434 162.0 2.16 2.84 350 3580 1,790 18.04 15.04 15
401 93.4 3.75 1.25 350 394 197 19.26 16.26 16
513 142.0 2.46 2.54 350 3093 1,547 18.38 15.38 15
441 16.2 5.00 - 81 3462 1,731 18.8 15.8 15
444 70.6 4.96 0.04 350 2871 1,436 17.49 14.49 14
44 41.4 5.00 - 207 3613 1,807 26.73 23.73 23
B5 - NTC1 NA - 5.00 NA 3253 1,627 26.72 23.72 23
B5 - NTC2 NA - 5.00 NA 783 392 28.34 25.34 23
Written on December 10, 2019