Quantstudio 3d master mix

Digital PCR (dPCR) was carried out by mixing 3.33 μl diluted template DNA (50 ng/μl, diluted 1:10 for E11 and E18, 1:2 for PN1 and undiluted for 3 M hippocampus); concentrations of DNA are described below and varied by experiment with 16.5 μl QuantStudio 3D master mix, 3.33 μl 20× gene expression assay, and 9 μl water [32.16 μl (enough for two chips with excess)] (Life Technologies). Reactions were loaded onto QuantStudio 3D digital PCR chips (20,000 wells per chip) using the QuantStudio 3D chip loader according to manufacturer’s instructions (Life Technologies). Chips were then sealed and cycled on a GeneAmp PCR system 9700 with a flatblock attachment using the following conditions: Stage 1—96 °C for 10 min, Stage 2—60 °C for 2 min and then 98 °C for 30 s (repeat Stage 2 39 times), Stage 3—60 °C for 2 min and an infinite 10 °C hold. Chips were then read in the QuantStudio 3D chip reader (Life Technologies) to obtain raw fluorescent values per well. Quality check of the chips and counting of positive and negative wells in order to determine copies/microliter were carried out on the QuantStudio 3D AnalysisSuite cloud software (LifeTechnologies) using the absolute quantification module and then calculated to copies per μg of input RNA including incorporating dilutions stated above.