TRIzol Reagent

RNA isolation / purification Tissue - Rat Spinal cord

Experiment
RNA isolation / purification Tissue - Rat Spinal cord
Product
TRIzol Reagent from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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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Discussion

Discussion

1 year ago

Author: Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Discussion

2 years ago

Author: Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Papers

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Manufacturer protocol

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