Purify and Fragment mRNA

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This step uses oligo(dT) magnetic beads to capture messenger RNAs (mRNAs) with polyA tails. The RNA is then fragmented and primed for complementary DNA (cDNA) synthesis.

• BBB (Bead Binding Buffer)
• BWB (Bead Washing Buffer)
• ELB (Elution Buffer)
• EPH3 (Elute, Prime, Fragment 3HC Mix)
• RPBX (RNA Purification Beads)
• Nuclease-free ultrapure water
• 1.7 ml microcentrifuge tube, RNase-free
• 96-well PCR plates, semiskirted (2)
• Microseal 'B' adhesive film
• Prepare for a later procedure:
– FSA (First Strand Synthesis Mix)
1. Prepare the following consumables:

Reagent

Storage

Instructions

BBB

2°C to 8°C

Vortex and invert to mix.

BWB

2°C to 8°C

Vortex and invert to mix.

ELB

2°C to 8°C

Vortex and invert to mix.

EPH3

-25°C to -15°C

Thaw at room temperature. Vortex to mix, and then centrifuge briefly.

FSA

-25°C to -15°C

Thaw on ice. Vortex to mix, and then centrifuge briefly.

RPBX

2°C to 8°C

Let stand for 10 minutes to bring to room temperature. Vortex and invert to mix.
2. Save the following mRNA_CAP program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 50 µl
• 65°C for 5 minutes
• 4°C for 30 seconds
• 23° for 5 minutes
• Hold at 23°C
3. Save the following mRNA_ELT program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 25 µl
• 80°C for 2 minutes
• Hold at 25°C
4. Save the following DEN94_8 program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Reaction volume is 19 µl
• 94°C for 8 minutes
• Hold at 4°C

Capture mRNA

1. In each well of a new PCR plate, dilute 25–1000 ng total RNA in nuclease-free ultrapure water to a volume of 25 µl.
2. Vortex RPBX to resuspend.
3. Add 25 µl RPBX to each well.
4. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Pipette 10 times, and then seal.
5. Place on the preprogrammed thermal cycler and run the mRNA_CAP program.

Total program time is ~15 minutes and each well contains a volume of 50 µl.

Elute mRNA

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Place on the magnetic stand and wait 2 minutes.
3. Remove and discard all supernatant.
4. Remove from the magnetic stand.
5. Add 100 µl BWB to each well.
6. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Pipette 10 times.
7. Place on the magnetic stand and wait 2 minutes.
8. Remove and discard all supernatant.
9. With a 20 µl pipette, remove all residual BWB.
10. Remove from the magnetic stand.
11. Add 25 µl ELB to each well.
12. Mix using either method:
• Seal and shake at 2200 rpm for 1 minute.
• Slowly pipette until the beads are resuspended, and then seal.
13. If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.
14. Centrifuge at 280 × g for 10 seconds.
15. Place on the preprogrammed thermal cycler and run the mRNA_ELT program.

Total program time is ~6 minutes and each well contains a volume of 25 µl.

Clean Up mRNA

1. In a 1.7 ml tube on ice, combine exactly the following volumes to prepare Fragmentation Master Mix. Multiply each volume by the number of samples.
• Nuclease-free ultrapure water (10.5 µl)
• EPH3 (10.5 µl)

Volumes include reagent overage for accurate pipetting.

2. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
3. Add 25 µl BBB to each well.
4. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Pipette 10 times.
5. Incubate at room temperature for 5 minutes.
6. Place on the magnetic stand and wait 2 minutes.
7. Remove and discard 50 µl supernatant.
8. Remove from the magnetic stand.
9. Add 100 µl BWB to each well.
10. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Pipette 10 times.
11. Place on the magnetic stand and wait 2 minutes.
12. Remove and discard all supernatant.
13. With a 20 µl pipette, remove all residual BWB.
14. Remove from the magnetic stand.
15. Thoroughly pipette Fragmentation Master Mix to mix.
16. Add 19 µl Fragmentation Master Mix to each well.
17. Mix using either method:
• Seal and shake at 2200 rpm for 1 minute.
• Slowly pipette until the beads are resuspended, and then seal.
18. If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.
19. Incubate at room temperature for 2 minutes.
20. Centrifuge at 280 × g for 10 seconds.

Fragment and Denature mRNA

1. Place on the preprogrammed thermal cycler and run the DEN94_8 program.

Total program time is ~10 minutes and each well contains a volume of 19 µl.

2. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
3. Place on the magnetic stand and wait 2 minutes.
4. Transfer 17 µl supernatant from each well to a new PCR plate.
5. Set aside the new PCR plate on ice.