QuantSeq Purification and qPCR Assay Batches 1 & 2

ds cDNA purification, pre-PCR

I purified the ds cDNA from batches 1 and 2. Notes on how to improve that process:

  • Bubbles! Bubbles make it difficult to use a mutichannel pipette. Need to improve handling to minimize bubble formation.
  • Should reduce amount of time I keep samples on magnet and to dry. The beads seem to crack a bit, and it’s difficult to resuspend them in the final elution step.
  • Max # of PCR plate columns for purification step = 8

qPCR assay for optimal endpoint PCR cycles

I performed the qPCR assay on all the ctenidia samples (batches 1 and 2). Here are the amplification curves:

image

Batch 1 end-point PCF cycle calculations

Sample No. 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
328 1,662 16.51 13.51 13
299 1,603 18.54 15.54 15
301 1,572 18.66 15.66 15
342 1,673 18.85 15.85 15
331 1,572 18.94 15.94 16
307 1,001 28.77 25.77 25
295 1,498 21.86 18.86 18
qPCR NTC 1,352 29.1 26.1 26
304 1,392 18.05 15.05 15
305 1,334 19.41 16.41 16
311 1,449 19.09 16.09 16
NTC2 - B1 1,521 25.92 22.92 23
298 1,437 18.27 15.27 15
348 1,394 19.33 16.33 16
315 1,496 18.74 15.74 15
qPCR NTC 1,483 28.23 25.23 25
344 1,545 18.19 15.19 15
325 2,029 17.64 14.64 14
338 1,572 19.31 16.31 16
347 1,547 17.9 14.9 15
312 1,510 18.77 15.77 15
321 1,561 18.03 15.03 15
333 1,651 18.07 15.07 15
291 1,265 18.06 15.06 15
308 1,297 19.1 16.1 16
NTC1 - B1 1,538 25.23 22.23 22
335 1,449 18.86 15.86 15
318 1,586 19.01 16.01 16
294 1,444 19.3 16.3 16
324 1,648 19.34 16.34 16

Batch 2 end-point PCR cycle calculations

Sample No. RFU @ endpoint 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
343 3583 1791.5 16.62 13.62 13
345 4129 2,065 17.31 14.31 14
303 3543 1,772 17.28 14.28 14
346 2823 1,412 17.75 14.75 14
302 3153 1,577 16.81 13.81 13
336 3300 1,650 22.82 19.82 19
292 3253 1,627 18.6 15.6 15
NTC1 -B2 3079 1,540 20.67 17.67 17
317 3576 1,788 18.8 15.8 15
322 2540 1,270 19.31 16.31 16
332 2689 1,345 20.02 17.02 17
334 2851 1,426 20.78 17.78 17
349 2825 1,413 18.57 15.57 15
337 3325 1,663 18.65 15.65 15
341 3152 1,576 17.61 14.61 14
313 3184 1,592 18.47 15.47 15
309 2962 1,481 18.05 15.05 14
327 2973 1,487 18.89 15.89 15
319 3037 1,519 20.88 17.88 16
326 2810 1,405 18.12 15.12 15
306 3698 1,849 21.19 18.19 18
323 3537 1,769 18.47 15.47 15
314 2996 1,498 20.58 17.58 17
316 2929 1,465 19.02 16.02 16
339 2761 1,381 19.33 16.33 16
293 2213 1,107 19.67 16.67 16
329 2164 1,082 18.72 15.72 15
296 2182 1,091 18.1 15.1 15

Notes

  • The highest quality RNA samples require 12 cycles. According to my QuantSeq rep, the very lowest quality and concentration RNA samples require 25 cycles.
  • I should use the optimal cycle number for end-point PCR, BUT some people that are in a hurry run all samples using the average of three consecutive cycles. For instance, if some samples need 14, some need 15, and some need 16, one can run all samples for 15 cycles. However, that is not the optimal protocol. I will proceed, but may need to re-generate a few libraries, which I will determine using Qubut and high sensitivity DNA chip on the Bioanalyzer.
Written on December 10, 2019