MagMAX RNA Isolation Protocol for Tempus Blood RNA Tubes

 

MagMAX for Stabilized Blood Tubes RNA Isolation Kit Protocol for Tempus Tubes (Cat. 4451893)

This is the manufacturer's protocol for purifying total RNA, including microRNA, from human whole blood collected in Tempus Blood RNA Tubes, paraphrased from the Applied Biosystems protocol (Part No. 4452007, Rev. A). It works with magnetic beads instead of spin columns, and you can run it in 1.5 mL tubes or in the 96-well Processing Plate supplied with the kit. Expect roughly 3 to 25 µg of RNA from 3 mL of blood.

Before you start

• Draw 3 mL of blood into the Tempus tube and shake or vortex it for 10 seconds straight away. Poor mixing means poor stabilization and microclots.
• Stabilized blood keeps for up to 5 days at 18 to 25 °C, up to 7 days at 4 °C, and long term at −20 to −80 °C (keep it off direct contact with dry ice).
• Prepare Wash Solution 1 by adding 8 mL of 100% isopropanol to each bottle of concentrate. Prepare Wash Solution 2 by adding 24 mL of 100% ethanol to the concentrate. Invert 3 to 4 times, mark the bottles, and store at room temperature. Each isolation uses 300 µL of each.
• You need a refrigerated swing-bucket centrifuge with 50 mL tube adapters that can reach 5,000 × g at 4 °C, a magnetic stand, and either a vortexer with a 1.5 mL tube adapter or an orbital plate shaker.
• Vortex adapter settings used by the manufacturer (scale of 1 to 10): 4 to 5 for binding and washes, 6 to 7 for digestion and elution. Orbital plate shaker settings: 6 to 7 for binding and washes, 8 to 9 for digestion, drying and elution. Test your own equipment with water so nothing spills.
• Wear gloves, use RNase-free tips, and wipe the bench and pipettes with an RNase decontamination solution.

Dilute the stabilized blood

1. If the sample is frozen, thaw the Tempus tube on ice for 10 to 30 minutes. Keep samples on ice as much as possible.
2. Pour the whole contents into a labeled 50 mL conical tube.
3. Add 3 mL of Tempus 1× PBS to reach 12 mL. If you collected less than 3 mL of blood, top up with PBS to 12 mL, otherwise yields drop sharply.
4. Cap tightly and vortex at vigorous speed for 30 seconds. Froth is normal. Go straight to the next section.

Pellet and wash the crude RNA

1. Centrifuge at 5,000 × g for 15 minutes at 4 °C. The pellet is transparent and invisible, so handle the tubes gently.
2. Pour off the supernatant.
3. Mix the Tempus Pre-Digestion Wash by gently inverting the bottle 3 to 4 times. Add 4 mLto each tube, recap, and vortex at moderate speed for 5 seconds, enough to draw the liquid about three-quarters of the way up the tube.
4. Centrifuge again at 5,000 × g for 10 minutes at 4 °C.
5. Decant the supernatant carefully. Decanting is recommended because a pipette can take the pellet with it.
6. Drain the tubes inverted on absorbent paper for 2 minutes, then blot the rims.
7. Keep the tubes on ice and move on immediately.

Resuspend the pellet and digest with protease and TURBO DNase

Just before use, prepare the resuspension mixture. Scale it by the number of samples and add 5% extra to cover pipetting loss.

Component	Volume for 1 sample
Tempus Resuspension Solution	117.5 µL
Tempus Proteinase	2.5 µL
Total	120 µL

1. Mix the components by vortexing at moderate speed, spin briefly for 2 to 3 seconds, and keep on ice.
2. Add 120 µL of the mixture to each conical tube, placing it at the bottom of the tube, and recap.
3. Vortex at gentle speed for 5 to 10 seconds to resuspend the pellet.
4. Transfer the full volume to a 1.5 mL microfuge tube or to one well of the 96-well Processing Plate. You can pause here and hold the resuspended pellets on ice for up to 4 hours (cover the plate wells).
5. Add 10 µL of TURBO DNase to each tube or well and mix at vigorous speed for 10 minutes. Do this at 18 to 25 °C. If your lab is colder than 18 °C, use a 25 °C incubator.

Bind the RNA to the beads

1. Vortex the RNA Binding Beads at vigorous speed until fully resuspended.
2. To each tube or well, add 50 µL of Binding Solution Concentrate and 20 µL of the bead suspension. Add the beads before the isopropanol.
3. Mix at moderate speed for 1 minute.
4. Add 200 µL of 100% isopropanol and mix at moderate speed for 3 minutes.
5. Capture the beads on the magnetic stand for about 1 to 3 minutes, until the solution clears.
6. Aspirate and discard all the supernatant without disturbing the beads.
7. Take the tubes or plate off the magnet. This is critical before the next step.

Wash twice with Wash Solution 1

1. Add 150 µL of Wash Solution 1 and mix at moderate speed for 1 minute.
2. Capture the beads for about 1 to 2 minutes and discard the supernatant.
3. Remove from the magnet, then repeat the wash once.

Wash twice with Wash Solution 2

1. Add 150 µL of Wash Solution 2 and mix at moderate speed for 1 minute.
2. Capture the beads for about 1 to 2 minutes and discard the supernatant.
3. Remove from the magnet, then repeat the wash once.

Dry the beads

• Tubes: open them and invert on absorbent paper for 2 minutes at room temperature. If liquid remains, remove it with a fine-tipped pipette without touching the beads, then invert for 1 minute more.
• Plate: shake at vigorous speed for 2 minutes at room temperature. If liquid remains, shake for 2 minutes more.
• Do not over-dry the beads, and make sure no alcohol is left before elution.

Elute the RNA

1. Add 80 µL of Elution Buffer (you can use anywhere from 20 to 80 µL depending on the concentration you want) and mix at vigorous speed for 4 minutes.
2. Capture the beads for about 1 to 3 minutes.
3. Transfer the supernatant to a nuclease-free tube without disturbing the beads. Keep it, because the purified RNA is in this supernatant.
4. Store on ice for immediate use, at −20 °C for up to 6 months, or at −80 °C for the long term.

Checking yield and quality

• Expect an A260/A280 ratio of 1.8 to 2.2. Values outside that range can still work for qRT-PCR.
• For human blood RNA from this kit, a 28S to 18S rRNA ratio of 1.0 or higher is considered acceptable. A RIN from a bioanalyzer is the more reliable measure of integrity.

Troubleshooting

• Low or no yield: check that the tube was filled to the black mark, that the blood was diluted to a final 12 mL with PBS and vortexed for 30 seconds, and that you used the full centrifuge times and speeds in a 50 mL tube in a swing-bucket rotor. Lysate that was exposed to more than 37 °C can be frozen, thawed and repurified. Also check that the beads were fully resuspended, were never frozen, and were added before the isopropanol.
• Lost pellet or beads: decant slowly rather than pipetting off the pellet, and aspirate slowly with the tip away from the captured beads.
• Beads clump during elution: warm the plate or tubes at 70 °C for 5 minutes and repeat the 4-minute mix. Next time, preheat the Elution Buffer to 70 to 80 °C. If the beads were over-dried, extend the elution mixing to 10 minutes.
• Bead carryover (light brown eluate): capture again on the magnet and move the RNA to a fresh tube. A small amount does not inhibit RT or RT-PCR.
• Too much DNA: make sure the digestion ran for the full 10 minutes at vigorous mixing and at 18 to 25 °C, and that the pellet was washed and drained well so stabilizing reagent was not carried over.
• RNA fails downstream: salt or alcohol carryover is the usual cause, so check that the correct alcohol and volume went into each wash concentrate, remove every supernatant fully, and let the beads dry before elution. A green or reddish eluate signals inhibitors, so spin it at 16,000 × g for 3 minutes and move the clear fraction to a new tube.

Source: Applied Biosystems, MagMAX for Stabilized Blood Tubes RNA Isolation Kit (Compatible with Tempus Blood RNA Tubes) Protocol, Part No. 4452007, Rev. A (2010).

October 26, 2026 Tags : MagMAX , MagMAX RNA Extraction , RNA extraction , RNA Isolation , Tempus Blood RNA Tubes , Tempus RNA Tubes

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