實(shí)驗(yàn)試劑

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1. 80% Ethanol

2. Isopropanol

3. Chloroform

實(shí)驗(yàn)設(shè)備

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1. Magnetic Stand for 1.5 ml tube (OBI # MSD-02)

2. Nuclease-free 1.5 and 2 ml centrifuge tubes

3. Microcentrifuge capable of 12,000 x g and 2-8°C

實(shí)驗(yàn)步驟

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Total RNA Isolation:

1. Homogenization and lysis of samples: follow either method below.

?? 1) Tissue Samples

Homogenize tissue samples in 1.5 mL of RNA-Solv ? Reagent per 100 mg of tissue using an appropriate mechanical homogenizer. Alternatively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 2 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (OBI Cat No. SS-1015-00). The sample volume should not exceed 10% of the volume of RNA-Solv ? Reagent used.

?? 2) Cells Grown in Suspension

Pellet cells by centrifugation. Lyse cells in RNA-Solv ? Reagent by repetitive pipetting. Use 1.5 mL of the RNA-Solv Reagent per 1 x 107 of animal, plant or yeast cells, or per 1 x 108 bacterial cells. Washing cells before addition of RNA-Solv ? Reagent should be avoided as this increases the possibility of mRNA degradation and RNase contamination

?? 3) Cells Grown in Monolayer

Lyse cells directly in a culture dish by adding 1.5 mL of RNA-Solv ? Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount of RNA-Solv ? Reagent added is based on the area of the culture dish (~1.5 mL per 10 cm2 ). An insufficient amount of RNA-Solv ? Reagent may result in contamination of the isolated RNA with DNA. Always use more RNA-Solv ? Reagent if in the lysate is too viscous to aspirate with a pipette.

2. Add 0.3 mL of chloroform per 1.5 mL of RNA-Solv ? Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it .

3. Centrifuge at 12,000 x g for 10 minutes at 4°C.

4. Precipitation of RNA. Transfer no more than 80% of the aqueous phase to a fresh 2 ml tube, and discard the lower organic phase. Precipitate the RNA from the aqueous phase by adding 750 ul of isopropanol per 1.5 mL of RNA-Solv ? Reagent used for the initial homogenization. Invert the sample 10-20 times. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes at 2-8°C.

5. Discard the supernatant and wash the RNA pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2-8°C.

6. Carefully aspirate and discard the ethanol. AIR DRY the RNA pellet for 5-10 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in redissolving RNA in water.

7. Add 200 ul RNase-Free water and vortex for 20 seconds. Incubate at room temperature to completely dissolve the RNA pellet.

Oligo(dT) Magnetic beads Washing Procedure:

8. Swirl or shake the vial of Oligo(dT) magnetic beads until the particles are in a homogeneous suspension.

9. Transfer 100 ul of Oligo(dT) Magnetic beads into a new tube. Place the tube on a magnetic separation device (MSD-02). The Oligo(dT) beads will migrate to the side of the tube nearest the magnet.

10. Remove the supernatant with a pipette while the tube remains on the magnet.

11. Remove the tube from the magnet and add 400 ul of mRNA Wash Buffer to resuspend the beads. Again place the tube on the magnet for 5 minutes.

12. Remove the supernatant while the tube remains on the magnet.

13. Remove the tube from the magnet and 200 ul of 2 x Binding Buffer to resuspend the beads.

mRNA Purification:

14. Incubate the purified RNA at 65°C for 3 minutes to disrupt secondary structures. Immediately place on ice for 3 minutes.

15. Transfer 200 ul of the RNA from step 14 into the tube containing the beads. Mix throughly and then place on a rotating mixer for 10 minutes at room temperature.

16. Place the tube on the magnet for 10 minutes. The liquid should be cleared after the magnetic beads are completely magnetized.

17. Aspirate the supernatant by pipetting. Remove the tube from magnetic stand.

18. Wash the magnetic beads by adding 300 μl mRNA Wash Buffer. Resuspend the magnetic beads by pipetting carefully a couple of times.

19. Collect the magnetic beads by place the tube on a magnetic separation device (MSD-02). Carefully remove all the supernatant.

20. Repeat the washing step as described in step 18-19.

mRNA Elution:

21. If the isolated mRNA does not need to be eluted off the beads, wash one more time using the same buffer that will be used in the downstream application, e.g. reverse transcription first strand synthesis buffer (without the enzyme). Then resuspend the beads in an appropriate volume of the downstream buffer.

22. If the isolated mRNA elution is required, add the desired amount (10-30 μl) of mRNA Elution Buffer (10mM Tris, 1mM EDTA). Heat to 65°C for 2 minutes and place the tube immediately on the magnet. Quickly transfer the eluted mRNA to a new RNA-Free tube.

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