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 |
|
|
|
|
|
|
|
|
|
|
|
|
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 |