Experiments and LOY analysis using digital droplet PCR (ddPCR)

JD Jan P. Dumanski
JH Jonatan Halvardson
HD Hanna Davies
ER Edyta Rychlicka-Buniowska
JM Jonas Mattisson
BM Behrooz Torabi Moghadam
NN Noemi Nagy
KW Kazimierz Węglarczyk
KB Karolina Bukowska-Strakova
MD Marcus Danielsson
PO Paweł Olszewski
AP Arkadiusz Piotrowski
EO Erin Oerton
AA Aleksandra Ambicka
MP Marcin Przewoźnik
ŁB Łukasz Bełch
TG Tomasz Grodzicki
PC Piotr L. Chłosta
SI Stefan Imreh
VG Vilmantas Giedraitis
LK Lena Kilander
JN Jessica Nordlund
AA Adam Ameur
UG Ulf Gyllensten
ÅJ Åsa Johansson
AJ Alicja Józkowicz
MS Maciej Siedlar
AK Alicja Klich-Rączka
JJ Janusz Jaszczyński
SE Stefan Enroth
JB Jarosław Baran
MI Martin Ingelsson
JP John R. B. Perry
JR Janusz Ryś
LF Lars A. Forsberg
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Additional quantification of the level of LOY for validation was performed using previously described procedure [21]. Briefly, extracted DNA was digested for 15 min with HindIII enzyme (Thermo Fischer) in 37 °C. After this, 50 ng of DNA was added together with PCR primers and probes targeting a known difference between the AMELX and AMELY gene assay C_990000001_10 (Thermo Fisher). Droplets were then generated using an automated droplet generator (Bio-Rad) and amplified using PCR. Droplets fluorescence intensity in two channels FAM and VIC was measured using the Bio-Rad’s QX200 Droplet Reader. The data were analyzed in Bio-Rad’s software QuantaSoft (version 1.7.4.0917) as described elsewhere [21]. The LOY quantification was performed by calculating the ratio between the amounts of AMELY and AMELX in each sample.

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