Veriquest probe one step qrt pcr master mix with rox

For qRT-PCR from brain tissue, dissections and RNA extraction were performed as described in Seigneur and Südhof (2017) (link). VeriQuest Probe One-Step qRT-PCR Master Mix with ROX (Affymetrix) and an ABI 7900HT Fast RT-PCR instrument (Applied Biosystems) were used. Samples were run in technical triplicates with Gapdh and Actb as loading controls. Data were normalized to the arithmetic mean of Gapdh and Actb, and mean 2^−ΔCt values are plotted.

RNA was isolated from DIV12 neurons that had been previously infected with lentivirus on DIV3–4 to allow Cre-mediated gene deletion or knockdown of ADAM10 using shRNA (see below). RNA was isolated using the PrepEase RNA spin kit according to the manufacturer’s instructions (Affymetrix). Nrxn1, Nrxn3, Lphn1, and Nlgn1 probes are described above. A prevalidated mouse ADAM10 assay (assay ID: Mm.PT.58.14225600) was obtained from Integrated DNA Technologies. For each reaction, 1 μl of RNA was run in triplicate using VeriQuest Probe one-step qRT-PCR master mix with ROX (Affymetrix). Reactions were loaded onto a QuantStudio3 Real-Time PCR machine (Thermo Fisher Scientific). The relative transcript level of the target mRNA in each sample was calculated as described above.