TRIzol Reagent

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	The dorsal horn of the spinal cord at the sacral and coccyx levels was homogenized in 1 ml of TRIzol reagent per 50 to 100 mg of tissue sample using TissueLyser (Qiagen). The samples were shaken at high speed in 2-ml round-bottomed microcentrifuge tubes with stainless steel beads for 15 min at room temperature. Following homogenization, the samples were centrifuged at 1200g for 5 min at 4°C, and the fatty layer was discarded. The cleared supernatant was transferred to a new microcentrifuge tube (Qiagen). For phase preparation, 200 μl of chloroform was added per 1 ml of TRIzol reagent, mixed for 15 s, and incubated for 2 min at room temperature. The mixture was then centrifuged at 13,300 rpm for 15 min at 4°C. The colorless upper aqueous phase containing RNA was transferred to a fresh tube and mixed with 600 μl of 70% molecular grade ethanol.

Protocol tips

The dorsal horn of the spinal cord at the sacral and coccyx levels was homogenized in 1 ml of TRIzol reagent per 50 to 100 mg of tissue sample using TissueLyser (Qiagen). The samples were shaken at high speed in 2-ml round-bottomed microcentrifuge tubes with stainless steel beads for 15 min at room temperature. Following homogenization, the samples were centrifuged at 1200g for 5 min at 4°C, and the fatty layer was discarded. The cleared supernatant was transferred to a new microcentrifuge tube (Qiagen). For phase preparation, 200 μl of chloroform was added per 1 ml of TRIzol reagent, mixed for 15 s, and incubated for 2 min at room temperature. The mixture was then centrifuged at 13,300 rpm for 15 min at 4°C. The colorless upper aqueous phase containing RNA was transferred to a fresh tube and mixed with 600 μl of 70% molecular grade ethanol.

Publication protocol

Left and right dorsal horns of the sacral segment of the spinal cord were dissected within 30 min after euthanasia, snap-frozen, and immediately placed at −80°C until further analysis. The dorsal horn of the spinal cord at the sacral and coccyx levels was homogenized in 1 ml of TRIzol reagent per 50 to 100 mg of tissue sample using TissueLyser (Qiagen). The samples were shaken at high speed in 2-ml round-bottomed microcentrifuge tubes with stainless steel beads for 15 min at room temperature. Following homogenization, the samples were centrifuged at 1200g for 5 min at 4°C, and the fatty layer was discarded. The cleared supernatant was transferred to a new microcentrifuge tube (Qiagen). For phase preparation, 200 μl of chloroform was added per 1 ml of TRIzol reagent, mixed for 15 s, and incubated for 2 min at room temperature. The mixture was then centrifuged at 13,300 rpm for 15 min at 4°C. The colorless upper aqueous phase containing RNA was transferred to a fresh tube and mixed with 600 μl of 70% molecular grade ethanol.

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