This experiment utilizes Ambion's MicroPoly(A)Pure kit to isolate mRNA. This experiment was derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
mRNA isolation assay utilizing Ambion's MicroPoly(A)Pure kit
Materials and Instruments
primary cell Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol
Tissue homogenizer MicroPoly(A)Pure kit
1. Buffers, solutions and reagents
Ethanol, 100%
2. Special equipment
Tissue homogenizer (optional), see II. Method steps 4
Water bath, preset to 70°C
3. Other
MicroPoly(A)Pure kit (Ambion)
4. Cells and Tissues
Primary Cells
II. Methods
1. Preheat the elution buffer to 70°C.
2. 250 g centrifuge to settle the cells and discard the supernatant.
3. Centrifuge after gentle resuspension in PBS and perform the first wash of the cell sediment. Discard the supernatant.
4. Add 250ul of lysate. Homogenize with a tissue homogenizer or by vigorous shaking or aspiration. Estimate the volume of lysis yield to use as the "starting volume".
5. Add 2x the starting volume of dilution buffer. Invert or shake for 10s to mix.
6. Centrifuge at 12000 g for 15 min at 4°C. Transfer the supernatant to a new tube.
7. Add a vial of oligo(dT) cellulose to the sample. Invert to mix. Incubate at room temperature with gentle stirring for 30-60 min.
8. Centrifuge at 4000 g for 3 min at room temperature.
9. Transfer the supernatant and keep the supernatant until the experiment proves that mRNA has been purified.
10. Add 1 ml of Binding Buffer to the oligodeoxythymidine cellulose precipitate, invert to mix, centrifuge at 4000 g for 3 min, discard Binding Buffer, and repeat this step twice.
11. Add 1 ml of Wash Buffer to the sediment, mix inverted, centrifuge at 4000 g for 3 min, remove and discard the Wash Buffer and repeat this step twice.
12. Place a centrifuge column in a wash tube and resuspend oligo(dT) cellulose in 400ul of Wash Buffer. Transfer the solution to a centrifuge column and centrifuge briefly at 4000 g. Discard the effluent.
13. Add 500ul of Wash Buffer to the centrifuge column. Mix the resin with a pipette tip, being careful not to damage the membrane in the column. Centrifuge briefly at room temperature, discard the supernatant and repeat the procedure. Save the last washed effluent.
14. Determine the A260 of the flow-through; if the A260 is less than 0.05, continue with the protocol; if the A260 is greater than 0.05, repeat with 500ul of Wash Buffer until the A260 is less than 0.05.
15. Place the centrifuge cartridge in a new microcentrifuge tube, add 100ul of Elution Buffer at 70°C to the cartridge, and immediately centrifuge at 5000 g. Add 100ul of Elution Buffer at 70°C to the cartridge. Add 100ul of hot Elution Buffer to the column and centrifuge briefly at 5000 g. Dispose of the column. Discard the column.
16. Add 20ul of 5mol/L ammonium acetate, 1ul of glycogen and 550ul of 100% ethanol to the eluted mRNA and precipitate at -20°C overnight.
17. Recover the mRNA by centrifugation at 12000 g or greater for 20 min at 4°C.
18. Carefully discard the supernatant, centrifuge again briefly, and remove all residual liquid with a tip pipette.
19. Re-dissolve the sediment in 1-20ul of DEPC-treated H2O/EDTA provided in the kit with vigorous shaking to completely dissolve the sediment. Store RNA at -70°C.