Isolation Of mRNA

mRNA was prepared using the protocol according to the mRNA isolation kit (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) as follows.

1. Incubate the elution buffer at 65°C, leave the lysis/binding buffer and wash buffer at room temperature.

2. Add ca. 1 mg total RNA sample to the Eppendorf tube, incubate the tubes at 65°C for 3 min and keep on ice.

3. Dilute up to 1 mg total RNA with at least 1 volume of lysis/binding buffer. The final volume should be 0.5-5 ml.

4. Add 25 ^l of oligo (dT) microbeads per 100 ^.g of the total RNA sample, and mix by pipetting. (Note: do not allow to bubble during the mixing).

5. Place a MACS Column Type M in the magnetic field of an appropriate MACS separator.

6. Prepare column by rinsing with 250 ^l of lysis/binding buffer and let buffer run through.

7. Apply total RNA sample on top of the column matrix. Let the solution pass through. Magnetically labeled mRNA is retained in the column.

8. Rinse the column with 1 x 250 ^l of lysis/binding buffer.

9. Rinse the column with 4 x 250 ^l of wash buffer.

10. Apply 200 ^l of pre-heated elution buffer on top of the column. mRNA is eluted by gravity. Typically, the third to sixth drop will contain around 90% of the isolated mRNA.

11. Add 1/10 volume of 3 M sodium acetate and 3 volume of ethanol to the eluted mRNA sample, and leave at -80°C for 1 h.

13. Decant the supernatant, wash the pellet with 75% ethanol and dry.

14. Dissolve in DEPC-treated distilled water and store at -80°C. The final mRNA concentration should be over 200 ng ^l-1.

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