Tempus Spin RNA Isolation Kit Protocol for Whole Blood RNA

 

Tempus Spin RNA Isolation Kit Protocol (Cat. 4380204)

This is the manufacturer's protocol for purifying RNA from blood collected in Tempus Blood RNA Tubes, paraphrased from the Thermo Fisher user guide (Pub. 4379232, Rev. E).

Before you start

• The protocol is designed for 3 mL of blood in a Tempus Blood RNA Tube, which contains 6 mL of stabilizing reagent.
• Immediately after collection, shake the tube vigorously or vortex it for 10 seconds. Poor mixing leads to microclots that can clog the filter.
• Stabilized blood keeps for up to 5 days at room temperature and 7 days at 4 °C, and it can be frozen at −20 °C or −80 °C.
• Keep samples on ice as much as possible during processing.

Isolate the RNA

1. If the tube is frozen, thaw it at room temperature (18 to 25 °C).
2. Pour the contents into a clean 50 mL conical tube.
3. Add 3 mL of Ca²⁺/Mg²⁺-free 1× PBS to bring the total to 12 mL. If you collected less than 3 mL of blood, top up with PBS to 12 mL. Skipping this dilution gives much lower yields.
4. Cap the tube tightly and vortex at maximum speed for 30 seconds. Less than 30 seconds can clog the column, and a layer of froth is normal.
5. Centrifuge at 4 °C at 3,000 × g for 30 minutes.
6. Carefully pour off the supernatant. The RNA pellet is transparent and invisible, so take care not to dislodge it.
7. Leave the tube inverted on absorbent paper for 1 to 2 minutes, then blot the rim.
8. Add 400 µL of RNA Purification Resuspension Solution, placing the pipette tip at the bottom of the tube so you do not wash blood residue down the walls. Vortex briefly to resuspend the pellet. The resuspended RNA can wait on ice.

Purify the RNA

Unless stated otherwise, every spin is 30 seconds at 16,000 × g.

1. Place a labeled purification filter in a waste collection tube and pre-wet the membrane with 100 µL of RNA Purification Wash Solution 1.
2. Load about 400 µL of the resuspended RNA onto the filter and spin.
3. Discard the flow-through, add 500 µL of Wash Solution 1, and spin.
4. Discard the flow-through, add 500 µL of Wash Solution 2, and spin.
5. Discard the flow-through and repeat the Wash Solution 2 step once more (500 µL, 30 seconds).
6. Discard the flow-through and spin for 30 seconds to dry the membrane.
7. Move the filter to a new labeled collection tube and add 100 µL of Nucleic Acid Purification Elution Solution. Incubate the whole tube at 70 °C for 2 minutes, then spin for 30 seconds.
8. Load about 100 µL of that eluate back onto the filter and spin for 2 minutes at 16,000 to 18,000 × g. No incubation is needed.
9. Discard the filter. Transfer about 90 µL of eluate to a fresh labeled tube, pipetting from the top of the liquid so the bottom 10 µL and any pellet stay undisturbed.
10. Store the RNA at −20 °C, or at −80 °C for the long term.

Troubleshooting

• If the filter stays black or brownish-red, the usual causes are poor mixing at collection or blood residue contaminating the sample. A repeat wash with 500 µL of Wash Solution 1followed by 500 µL of Wash Solution 2 can help.
• Degraded RNA is blamed on residual protein with RNase activity, or on the lysate being exposed to temperatures above 37 °C. For the first cause, add more wash steps until the membrane looks white.
• Expected yield is 6 to 25 µg of RNA from 3 mL of blood, with an A260/280 ratio above 1.9.

Source: Applied Biosystems, Tempus Blood RNA Tube and Tempus Spin RNA Isolation Kit User Guide, Pub. 4379232, Rev. E (2018).

October 26, 2026 Tags : Blood RNA , Protocols , RNA extraction , Tempus Spin RNA Kit

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