Extraction of Total RNA

Extraction of Total RNA

① Collect cells: Collect the splenic cells after removing the supernatant (IL-2), centrifuge at 300 rpm at room temperature for 5 minutes, and completely remove the supernatant. Adjust the cell concentration to 5×10⁶ cells/mL using PBS solution.

② Lyse the cells: Add 350 μL of RLT Solution to the sample according to the kit instructions, and shake vigorously to mix.

③ Homogenize with a homogenizer for 30 seconds.

④ Add an equal volume of 70% ethanol, and mix with a pipette tip.

⑤ Place the UNIQ-10 column in a 2 mL collection tube, add 700 μL of the sample to the UNIQ-10 column, and centrifuge at 8000 rpm at room temperature for 1 minute.

⑥ Discard the waste liquid in the collection tube, place the UNIQ-10 column back in the same collection tube, add 500 μL of RW Solution to the column, let it sit for 1 minute at room temperature, and centrifuge at 10000 rpm for 30 seconds. Discard the waste liquid in the collection tube, and place the column back in the same collection tube.

⑦ Add 500 μL of diluted RPE Solution to the UNIQ-10 column, centrifuge at 10000 rpm at room temperature for 1 minute. Discard the waste liquid in the collection tube, and place the column back in the same collection tube.

⑧ Add 500 μL of diluted RPE Solution to the UNIQ-10 column, centrifuge at 10000 rpm at room temperature for 1 minute. Discard the waste liquid in the collection tube, and place the column back in the same collection tube.

⑨ Centrifuge at 10000 rpm at room temperature for 1 minute to remove any remaining RPE Solution. Place the UNIQ-10 column in an RNase-free Eppendorf tube, draw 50 μL of DEPC-H₂O and add it to the center of the column membrane, leave it at 50°C for 2 minutes, then centrifuge at 10000 rpm at room temperature for 1 minute. The liquid in the Eppendorf tube is the extracted total RNA.