Preparation Before Assay

▓ Add 10 μl ß-ME to 1 mL of RL Buffer.

Note: Prepared RL Buffer can be stored at room temperature for up to 1 month.

【Whole Blood】

Sample Preparation

① Add 400 μl of human whole blood with 2 ml of RBC Lysis Buffer in a > 3ml RNase-free microcentrifuge tube, and mix completely by inversion. Do not vortex.

Note: For optimal results, the volume of the mixture (blood + RBC Lysis Buffer) should not exceed 4/5 of the volume of the tube to allow efficient mixing.

② Incubate on ice for 10 minutes and invert 3 times during incubation.

③ Centrifuge at 4°C at 2,500 rpm (500 x g) for 5 minutes, and discard the supernatant completely.

④ Add 400 μl of RBC Lysis Buffer to the cell pellet. Resuspend cells by vortex briefly.

⑤ Incubate on ice for 3 minutes.

⑥ Centrifuge at 4°C at 2,500 rpm (500 x g) for 5 minutes, and discard the supernatant completely.

⑦ Add 400 μl of RL Buffer (ß-ME added) to the cell pellet and mix by vortexing.

⑧ Incubate at room temperature for 5 minutes.

【Cell】

Sample Preparation

① Transfer the max. 5 x 10⁶ of cells to a microcentrifuge tube.

② Centrifuge at 4°C at 2,500 rpm (500 x g) for 5 minutes, and discard the supernatant completely.

③ Add 400 μl of RL Buffer (ß-ME added) and mix by vortexing.

④ Incubate at room temperature for 5 minutes.

Assay Procedures

① Add 400 μl of RB Buffer to the sample and mix by pipetting immediately for 10 seconds.

② Transfer 450 μl of the mixture to Nanosep®.

③ Centrifuge at 13,000 rpm (10,000 x g) for 3 minutes.

④ Remove the retentate cup of Nanosep®, discard the filtrate from the filtrate tube, and then place the retentate cup back into the filtrate tube of Nanosep®.

⑤ Transfer the remaining mixture to Nanosep®.

⑥ Repeat ③ and ④.

Optional: Perform DNA Elimination Procedures between ⑥and ⑦if needed.

⑦ Add 450 μl of RW1 Buffer to Nanosep®.

⑧ Centrifuge at 13,000 rpm (10,000 x g) for 1 minute.

⑨ Remove the retentate cup of Nanosep®, discard the filtrate from the filtrate tube, and then place the retentate cup back into the filtrate tube of Nanosep®.

⑩ Add 450 μl of RW2 Buffer to Nanosep®.

⑪ Centrifuge at 13,000 rpm (10,000 x g) for 1 minute.

⑫ Remove the retentate cup of Nanosep®, discard the filtrate from the filtrate tube, and then place the retentate cup back into the filtrate tube of Nanosep®.

⑬ Repeat ⑩, ⑪ and ⑫.

⑭ Centrifuge at 13,000 rpm (10,000 x g) for 3 minutes to dry NAB Filter.

⑮ Remove the retentate cup of Nanosep®, and transfer it into the new filtrate tube.

⑯ Add 50 μl of RNase-Free Water into the CENTER of the retentate cup.

⑰ Let the device stand for at least 2 minutes so NAB Filter can be soaked completely.

⑱ Centrifuge at 13,000 rpm (10,000 x g) for 2 minutes to elute the purified RNA.