The 380-replicates set is a dilution consisting of 150,000 molecules (see the Admonition section below) of the MYCN oligonucleotide was created in 10 ng/µl yeast tRNA carrier. qPCR amplifications were performed in 380 replicated 8 µl reactions and quadruplicated reactions of the NTC sample were performed on the same 384-well plate.
qPCR was done on a CFX 384 instrument (Bio-Rad). A 384-well qPCR plate was prepared using a 96-well head pipetting robot (Tecan Freedom Evo 150). qPCR amplifications were performed in 8 µl containing:
4 µl iQ SYBR Green Supermix (Bio-Rad);
0.4 µl forward and 0.4 µl reverse primer (5 µM each);
0.2 µl nuclease-free water;
3 µl of standard oligonucleotide.
All reactions were performed in 384-well plates (Hard-Shell 384-well microplates and Microseal B clear using adhesive seals (Bio-Rad).
The cycling conditions were comprised of 3 min polymerase activation at 95 °C and 45 cycles of 15 s at 95 °C and 30 s at 60 °C followed by a dissociation curve analysis from 60 to 95 °C.
Admonition
The concentration information for the curves in this data set was seemingly incorrectly indicated in the original publication (confirmed by email with the senior author Prof. Jo Vandesompele). Instead of the 15,000 copies reported in the original paper, the number of copies was most likely 150,000 copies (10x the value indicated).
Format
ds_380
A data frame with 17,280 rows and 9 columns:
wellWell identifier.
replicateReplicate identifier.
dyeIn all reactions the SYBR Green I master mix (Roche) was used, so the value is always
"SYBR".targetTarget identifier: MYCN.
sample_typeSample type:
"ntc"(no template control) or"std"(standard).copiesStandard copy number: either
0(for NTCs), or150000for standards.dilutionDilution factor (the reciprocal of
copies). Higher number means greater dilution.cyclePCR cycle.
fluorRaw fluorescence values.