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    RNA Purification Protocol for 100-200 mg of Animal Tissue

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    实验原理

     

    E.Z.N.A.® SQ Tissue RNA uses a highly efficient solution based system to provide a convenient, fast, reliable and non-toxic method to isolate high quality RNA from various samples. Samples are first lysed with RCL buffer. Cellular proteins and genomic DNA are removed by precipitation with PNP Buffer while RNA will remain in solution. RNA is further purified by isopropanol precipitation.

    实验试剂

     

    1. Isopropanol

    2. 70% ethanol

    实验设备

     

    1. Centrifuge capable of 3,000-5,000 x g

    2. High speed centrifuge tube with capacity of at least 15 ml

    3. Ice bath

    4. Ground pestle

    实验步骤

     

    1. Dissect tissue quickly and freeze in liquid nitrogen. Storage at -70°C. Fresh tissue can also be used. Work quickly and keep the sample on ice at all times.

    2. Add 100-200 mg frozen ground tissue or fresh tissue to a 15 mL centrifuge tube containing 6 ml RCL Buffer. Homogenize the sample using 5-10 strokes with tube pestle.

    3. Add 2 ml of PNP Buffer to the cell lysate. Mix the sample gently by inverting the tube 10 times.

    4. Place the tube on ice for 5 minutes.

    5. Centrifuge at 3,000-5,000 g for 10 minutes at room temperature. The precipitated protein and DNA will form a tight pellet.

    6. Transfer the supernatant to a new 15 ml sterile centrifuge tube that containing 6 ml of 100% isopropanol.

    7. Gently mix the solution by inverting the tube 30-40 times.

    8. Centrifuge at 3,000-5,000 x g for 15 minutes at room temperature.

    9. Pour of the supernatant and drain the tube briefly on a clean absorbent paper. Add 6 ml of 70% ethanol and invert the tube few times to wash the RNA pellet.

    10. Centrifuge at 3,000-5,000 x g for 10 minutes at room temperature. Carefully pour off the ethanol. Pellet may be very loose at this point so pour slowly and watch the pellet.

    11. Invert the tube on a clean adsorbent paper and air dry the pellet for 10-15 minutes.

    12. Add 250 ul of DEPC Water and vortex for 1 minutes to mix.

    13. Incubate sample on ice for at least 30 minutes. Vortex sample vigorously for 10 seconds and pulse spin.

    14. Store RNA at -70°C.

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