Digital Droplet PCR
mRNA copy number per nanogram of Total RNA was determined by one-step
reverse transcription digital droplet PCR (RT-ddPCR). Primers were
designed to amplify a 150 bp region in the middle of eGFP, universal to
every construct under investigation. ddPCR reaction mixtures of a final
volume of 20 ul were comprised of 5 ul one step RT-ddPCR supermix
(Bio-Rad), 2 ul reverse transcriptase (Bio-Rad), 1 ul 300 mM DTT
(Bio-Rad), 1 ul 900 nM forward/reverse primer mix (Integrated DNA
Technologies), 1 ul 250 nM 5’-FAM probe (Integrated DNA Technologies),
9ul H20 and 1 ul Total RNA at a concentration of 1 ng/ul. The 20ul
reaction mixture and 70ul of droplet generation oil (Bio–Rad) were
loaded into a DG8 Cartridge, and 40ul of droplets were generated with
the Bio-Rad QX200 droplet generator. Droplets were transferred to a
96-well PCR plate (Bio-Rad), sealed with foil, and placed in a Bio-rad
C1000 thermal cycler. Reverse transcription was performed at 50C for 1
hour. Polymerase activation was carried out for 10 minutes at 95C,
before 40 cycles of denaturation for 1 minute at 95C, before a combined
annealing and extension phase for 1 minute at 60C. Enzymes were then
deactivated at 98C for 10 minutes, before a final hold phase at 12C for
30 minutes. Positive droplets were detected by the QX200 droplet reader
(Bio-Rad), using automatically assigned amplitude thresholds determined
by QuantaSoft software (Bio-Rad). Samples were only used in analysis if
the number of measured droplets exceeded 12000.