Isolation of mRNA from Mammalian Cells

Step 1
•    Aliquot the appropriate volume of Oligo d(T)25 Magnetic Beads for the scale of isolation (Table 1).
Add 200 µl of Lysis/Binding Buffer to beads, vortex briefly and mix with agitation for 2 minutes. Beads should remain in the lysis/binding wash solution until removal immediately before adding the cell lysate.

Step 2
•    Adherent Cells
1. Aspirate media from cell culture plate and wash once with cold sterile 1X PBS (pH 7.4).
2. Add the appropriate volume of Lysis/Binding Buffer (see Table 1) to cells and gently swirl by hand.

•    Suspension Cells
1. Pellet Cells by centrifuging at 1,000 rpm for 5 minutes at 4°C.
2. Aspirate media and wash once with cold sterile 1X PBS (pH 7.4).
3. Pellet again, discard PBS and add the appropriate volume of Lysis/Binding Buffer (Table 1) to cells.
4. Agitate to suspend cells in Lysis/Binding Buffer.

Step 3
•    Incubate at RT for 5 minutes with gentle agitation. If the solution is viscous, pass the lysate several times through a 21-gauge needle attached to a 1 or 2-ml syringe. A noticeable decrease in viscosity should be observed.
•    Place the microcentrifuge tube containing the beads and lysis/binding wash into the magnetic rack and pull the magnetic beads to the side of the tube.

Step 4
•    Remove Lysis/Binding wash and add the cell lysate to the equilibrated magnetic beads.
•    Place cell lysate-and-bead suspension on the agitator and incubate at RT for 10 minutes.
•    Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard supernatant.

Step 5
•    Add the appropriate volume of Wash Buffer 1 (see Table 1) to the beads and mix with agitation for 1 minute.
•    Place microcentrifuge tube into magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
•    Repeat Step 5, once.

Step 6
•    Add the appropriate volume of Wash Buffer 2 (see Table 1) to the beads and mix with agitation for 1 minute.
•    Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
•    Repeat Step 6, once.

Step 7
•    Add the appropriate volume of Low Salt Buffer (Table 1) to the beads and mix with agitation for 1 minute.
•    Place microcentrifuge tube in the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.

Step 8
•    Add the appropriate volume of Elution Buffer (Table 1) and vortex gently to suspend beads.
•    Incubate at 50°C for 2 minutes with occasional agitation to elute poly(A)+ RNA. This step enables > 90% of the poly(A)+ RNA bound to the beads to be recovered.
•    Place microcentrifuge tube in the magnetic rack and pull the magnetic beads to the side of the tube. Transfer eluent to a clean, sterile RNase-free tube and store on ice for immediate quantitation or place at –80°C for long term storage.

Quantitation of Isolated poly(A)+ RNA:
Quantitation of isolated poly(A)+ RNA can be determined by measurement of A260 nm using a
Nanodrop™ spectrophotometer. The A260/280 ratio should be 1.6 or greater. The amount of isolated RNA will vary with the source of the RNA sample. For RNA 1A 260 Unit = ~ 40 μg /ml.

Notes: The eluted mRNA should be placed in the magnetic rack while removing aliquots for quantitation to avoid pipetting of beads, which will interfere with spectrophotometric analysis. Isolated poly(A)+ RNA is usually greater than 70% pure. Ribosomal RNA (rRNA) and trace amounts of genomic DNA (gDNA) are sometimes seen as impurities in isolated poly(A)+ RNA. The presence of rRNA will not interfere with the analysis of results from Northern Blotting and RT-PCR experiments. Trace gDNA contamination is usually low enough to permit valid qRT-PCR quantitation of mRNA transcripts without further purification.

Reuse of Oligo d(T)25 beads:
•    Oligo d(T)25 beads can be re-used for a second-round of purification of a poly(A)+ RNA eluent without regeneration. Wash beads once with an additional 100 µl of elution buffer. Place in magnetic rack and pull beads to the side of the tube. Remove and discard wash solution. Wash beads once with 200 μl of Lysis/Binding Buffer then re-apply previously isolated eluent to beads after adjusting salt concentration to 0.5 M LiCl or 0.5 M NaCl. Repeat isolation steps.

Regeneration of Oligo d(T)25 beads:

•    Oligo d(T)25 beads can be regenerated and used to isolate RNA from a different source. Add 0.1 N NaOH to the beads and incubate at room temperature with agitation for 5 minutes. Wash, equilibrate beads and store beads in sterile RNase-free 1X PBS (pH 7.4) containing 0.1% Tween 20.

Troubleshooting:
•    Viscous lysate
Certain cells lines and types of tissue will produce lysates that are more viscous than normal. Pass these cell or tissue lysates several times through a 21-gauge needle. If sample lysate remains viscous after additional shearing, proceed with isolation. Poly(A)+ RNA can be isolated under these conditions but is more likely to be contaminated with gDNA.

•    Clumping of beads
Clumping of beads is usually caused by non-sheared gDNA. If lysis- bead- suspension clumps, rapidly pipette cell or tissue lysate up and down with a 1 ml pipetman to further reduce viscosity. Poly(A)+ RNA can still be isolated under these conditions but is more likely to be contaminated with gDNA.

•    Genomic DNA contamination
Lower the ratio of cells or tissue to Lysis/Binding Buffer volume. Increase the binding incubation time if the lysate remains viscous after additional shearing. If intended downstream application requires mRNA be gDNA free, a second round of isolation should be done. See reuse of Oligo d(T)25 Magnetic Beads.

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