RNA isolation and quantitative real-time PCR (qRT-PCR)
For the gene expression analysis, animals treated with the lower doses
of the drugs were used, as these correspond to the therapeutic doses of
these agents in the treatment of major depressive disorder and the doses
of duloxetine in the treatment of OA pain (see Discussion for further
dosing information) [21]. Knee joint, DRG and spinal cord tissues
were homogenized in Tissue Lyser II (Qiagen) by using stainless steel
beads. Total RNA was extracted by using Trizol reagent (Thermo Fisher
Scientific) and isolated RNA was additionally treated with RapidOut DNA
Removal Kit according to the manufacturer′s protocol (Thermo Fisher
Scientific). RevertAid Reverse Transcriptase (Thermo Fisher Scientific)
with random hexamers (Thermo Fisher Scientific) and RiboLock RNase
inhibitor (Thermo Fisher Scientific) were used to transcribe 0.5 µg of
RNA as a template. Quantitative real-time PCR was performed with IC
Green qPCR Universal Kit (NIPPON Genetics, Düren, Germany) under the
following conditions: 2 min at 95°C activation, 40 cycles of 5 s at 95°C
and 30 s at 60°C in Line-Gene 9600 Plus Real-Time PCR (Hangzhou Bioer
Technology). All results are normalized to the housekeeping Gapdhgene and expressed as relative target abundance by using the 2-ΔΔCt
method [22]. All primers for Ngf , Il-1β , Tnf-α ,Bdnf and Tac1 (Tachykinin 1 - a gene that encodes
substance P) [23] and Gapdh gene expression analysis are
listed in Supplementary Table 1. Primers were purchased from Thermo
Fisher Scientific.