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Modified Total RNA Protocol for Heart, Muscle, Brain and Skin tissue

互联网2013-11-13

647

实验步骤

Note: Always use less starting material (<500mg) for the first time isolation to ensure a good result. If results obtained are satisfactory increase amount of starting material.

1. Disrupt the tissue and homogenize lysate in 6.0mL TRK lysis buffer with a rotorstator homogenizer. Transfer the homogenate to a 50ml centrifuge tube which is capable of 3500 x g.

2. Add 15ml ddH₂ O to the homogenate and add 220 ul Proteinase K or OB Protease (20mg/ml). Vortex for 30 seconds to mix. Incubate at 60°C for 15- 30 minutes.

3. Centrifuge at 3500 x g for 10 minutes and transfer clear supernatant (around 22.0ml) to a new 50 ml tube. (Avoid any precipitate or film on top of the supernatant may formed during process of step 2.)

4. Add 0.5 volume of absolute ethanol (11ml) to the sample and mix with pipetting.

5. Transfer 13ml of the mixture including any precipitate may have formed in step 4 to the HiBind^® RNA Maxi-column placed in a 50ml centrifuge tube (supplied). Closed the tube and centrifuge at 3500-5000 x g for 10 minutes. Discard the flow-through. Re-use the tube for next step.

6. Repeat step 5 twice until all the mixture from step 4 pass through the HiBind^® RNA Maxi-column.

7. Add 15 ml RNA Wash Buffer I, cap the 50 ml collecting tube and centrifuge for 5 min at 3,500-5,000 x g at room temperature. Discard flow-through liquid and reuse 50 ml tube in step 8.

8. Add 15 ml RNA Wash Buffer II, cap the 50 ml collecting tube and centrifuge for 5 min at 3,500-5,000 x g at room temperature. Again, discard flow-through liquid and reuse 50 ml tube in step 9.

9. Repeat step 8 with another 15 ml RNA Wash Buffer II. Discard flow-through liquid and re-insert the spin column in the empty 50 ml collecting tube. Centrifuge the empty column for 10 min at 5,000 x g to dry the HiBind^® matrix. This step is critical for removing traces of ethanol that may otherwise interfere with subsequent downstream applications.

Note: When a vacuum oven is available, place the maxi column into a vacuum oven which is preset at 60°C for 10-15 minutes. This will ensure that the column can be completely dried before elution.

10. RNA elution: transfer the HiBind^® RNA maxi column to a new 50 ml centrifuge tube (not supplied) and pipet 850 ul DEPC-treated water directly onto the matrix. For expected RNA yields greater than 1 mg use 1.25 ml of water. Cap the tube and allow the matrix to soak for 1 min. Centrifuge for 5 min at 5,000 x g.

Tip: Yield will be increased by 30%-70% by repeating the elution with a second volume of DEPC-treated water. For a higher final RNA concentration, this second elution may be performed using the first eluate. However, this will result in a ~30% lower overall yield.

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