The cells were collected, washed with phosphate-buffered saline, and lysed by the specific lysis buffer provided by the kit.
Cells were harvested and homogenized with the lysis buffer provided by the kit at 4°C.
Freshly collected leaves were pooled (up to 6 individuals) and ground in a mortar and pestle in demineralised water or extraction buffer provided by the manufacturer of the test strips used (see hereafter).
For the elution, water was used instead of the buffer provided by the kit.
For BDNF measurements, the plasma samples were diluted 1∶5 in block and sample buffer provided by the kit.
The incubation was done in the T4 poly nucleotide kinase reaction buffer provided by the supplier (70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT, pH 7.6 at 25°C) for 30 min at 37°C.
Ten microliters of each sample were amplified by PCR utilizing one unit of Taq polymerase (PlatinumTaq Invitrogen, Milan, Italy) in 50 µl of the buffer provided by the manufacturer with 3 mM MgCl2, and 2.5 mM of each dNTP.
Total RNA, including miRNA, was extracted from 50 µl of serum using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions, and finally eluted with 50 µl of elution buffer provided by the manufacturer.
20 50 ng bisulfite treated DNA were amplified using 0.5 units of Taq polymerase (Platinium Tag, Invitrogen, Karlsruhe Germany) in the presence of 200 µM dNTPs, 1.5 mM MgCl2 and 10 pmol of each primer in the reaction buffer provided by the manufacturer in a final volume of 25 µl.
Sera were diluted 1 50 using the dilution buffer provided by the manufacturer.
Briefly, serum samples (50 μl) were diluted 1 10 in assay buffer provided by the manufacturer.
