Oligo dt16 primer

The animals were sacrificed and L4–6 spinal cord segments were collected in tubes with RNAlater (Qiagen Inc., Valencia, CA, United States) and stored at −80°C until RNA isolation. Total RNA was isolated from these tissues according to Chomczynski’s method (Chomczynski and Sacchi, 1987 (link)) and reverse transcribed using Omniscript reverse transcriptase (Qiagen Inc., Valencia, CA, United States) at 37°C for 60 min. The reaction was performed in the presence of the RNase inhibitor rRNAsin (Promega, Madison, WI, United States) and an oligo (dT16) primer (Qiagen) to selectively amplify the mRNA. For quantitative PCR, 45 ng of cDNA was used as a template. Reactions were performed using Assay-On-Demand TaqMan probes and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA, United States) according to the manufacturer’s protocol. Reactions were run on a Real-Time PCR iCycler IQ (Bio-Rad, Hercules, CA, United States) with software version 3.0. The expression levels of Kcc2 were normalized to ß-actin.

Tissue from ipsilateral lumbar segments of the spinal cord (L4–L6) was collected immediately after decapitation from naive and CCI-exposed mice on days 2, 7, and 14 after sciatic nerve injury for quantitative real-time PCR (RT-qPCR) analysis. Total RNA extraction was performed using TRIzol reagent (Invitrogen, Carlsbad, USA) on the basis of a previously described protocol (36 (link)). The concentration and quality of RNA were measured by a DeNovix DS-11 Spectrophotometer (DeNovix Inc., Wilmington, USA). The Omniscript RT Kit (Qiagen Inc., Hilden, Germany), oligo (dT16) primer (Qiagen Inc., Hilden, Germany), and RNAse inhibitor (rRNasin, Promega, Mannheim, Germany) were used for reverse transcription of 1 μg of total RNA. The obtained cDNA was diluted 1:10 with RNase-/DNase-free H₂O. RT-qPCR was conducted with ~50 ng of cDNA from each sample using Assay-On-Demand TaqMan probes (Applied Biosystems, Foster City, USA) on an iCycler device (Bio-Rad, Hercules, Warsaw, Poland). The following TaqMan primers were used: Mm03024075_m1 (hypoxanthine-guanine phosphoribosyltransferase, HPRT); Mm01244826_g1 (CCL17); Mm00436439_m1 (CCL22); and Mm00441243_g1 (CCL2). Based on our previous study (25 (link)), HPRT was used as an endogenous control and an adequate housekeeping gene. There were no significant changes in HPRT expression between groups.

Reverse transcription was performed on 2 μg of total RNA using Omniscript reverse transcriptase (Qiagen Inc.) at 37°C for 60 min. RT reactions were carried out in the presence of an RNAse inhibitor (rRNAsin, Promega) and an oligo (dT16) primer (Qiagen Inc.). cDNA was diluted 1:10 with H₂O, and for each reaction, ~ 50 ng of cDNA synthesized from the total RNA of an individual animal was used for the quantitative real-time PCR (qPCR) reaction. qPCR was performed using Assay-On-Demand TaqMan probes according to the manufacturer’s protocol (Applied Biosystems), and the reactions were run on an iCycler device (BioRad, Hercules). The following TaqMan primers and probes were used: Rn01527838_g1 (Hprt, hypoxanthine guanine rat hypoxanthine guanine phosphoribosyl transferase); Rn01522736_m1 (Rwdd3); Rn01772952_m1 (Fam22f); Rn00580189_m1 (Slc7a7); Rn01423590_m1 (Cd40); Rn00595553_m1 (Gimap5); Rn01505455_m1 (Apobec3b); and Rn01459401_m1 (Clec7a). The amplification efficiency for each assay (between 1.7 and 2) was determined by running a standard dilution curve. The cycle threshold values were calculated automatically by the iCycler IQ 3.0 software using the default parameters. RNA abundance was calculated as 2^{-(threshold cycle)}. HPRT transcript levels do not significantly change in rats exposed to CCI [15 (link)] and, therefore, served as an adequate housekeeping gene.