Ambion trizol

Cells were lysed using 1 ml Ambion TRIzol (#15596018, Invitrogen, Carlsbad, USA) per 6-well according to the manufacturer’s protocol, the resulting RNA phase was washed and DNA digested using the RNeasy Mini Kit (#74104 and #79254, Qiagen, Hilden Germany), 1 μg/μl of cDNA was synthesized with the iScript cDNA synthesis Kit (#1708890, BioRad, Feldkirchen, Germany), the resulting cDNA was used undiluted for dPCR-based detection of the OXTR transcript with a Qiacuity dPCR cycler (Qiagen) in an 8.5 k 24-well Qiacuity Nanoplate and QuantiNova LNA probe PCR assays (see Table 1). Homo sapiens ACTB was used as a reference gene to control for even cDNA content. Cycling conditions were as follows: 2 min at 95°C heat activation, 15 s 95°C denaturation, 30 s 60°C annealing/extension for 40 cycles. Imaging was set to 500 ms exposure and gain of 20.

Total RNA was extracted from up to 100 mg of frozen samples using the RNeasy Plant Mini Kit (Qiagen, Germany) and immediately stored at −20 °C. DNase I (Qiagen, Germany) was used to avoid genomic DNA contamination. Total RNA from flag leaves was isolated by using Ambion TRIzol (Invitrogen, USA) and then purified by using a Qiagen clean-up kit (Qiagen, Germany). The quality and integrity of the isolated RNA were determined by electrophoresis on 1 % (w/v) agarose gels. The concentration was assessed using a NanoDrop-1000 spectrophotometer (Nanodrop Technologies Inc., USA).
For cDNA synthesis for the first and second biological replicates, 1 μg of total RNA, 50 U Expand Reverse Transcriptase (Roche, Germany), 50 pmoles of oligo (dT) primers and 100 pmoles of random hexamer (pdN6) primers were used in a 20 μL reverse transcription reaction. The final reaction mix was incubated at 42 °C for 1 h. For the third biological replicate, the reverse transcription reaction was carried out using cDNA synthesis kit (Clontech, Japan). The cDNA products were diluted 10-fold with Milli-Q water and stored at −20 °C.

RNA from cells was extracted in Trizol Ambion (AM9738, ThermoFisher, Madrid, Spain) following the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000 spectrophotometer (ThermoFisher), and 1 µg of RNA was reverse-transcribed to cDNA with a Transcriptor First-Strand cDNA Synthesis kit (04379012001, Roche). qPCR assay was carried out with 5 µL of this template cDNA, 10 µL of the SYBR Green PCR Master Mix cocktail (4309155, ThermoFisher) and 250 nM forward and reverse primers (Supplementary Table S2). RPLP0 (36B4) was chosen as a housekeeping endogenous control for normalization purposes. A qPCR reaction was carried out in MyIQ RealTime PCR System (BioRad, Madrid, Spain). Result analysis was conducted with the IQ5 program (BioRad) following the 2^−ΔΔCt method.