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        RNA Isolation From Animal tissue or cell culture

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        745

        实验试剂

         

        Regents supplied by user:

        1. 96-100% ethanol

        2. $-Mercaptoethanol

        实验设备

         

        Equipments supplied by user:

        1. RNase-free filter pipette tips

        2. Microcentrifuge capable of 12,000 x g

        实验步骤

         

        1. Determine the starting amount of sample. Do not use more than 5 x 105 cells or 5 mg tissue.

        2. Lyse cells (< 5x 105 ) or tissues (< 5mg) with 350 ul of TRK Lysis Buffer. Remember to add 20 ul of 2-mercaptoethanol per 1 mL of TRK Buffer before use.

        3. Disrupt the tissue or cells and Homogenize the lysate in TRK Lysis Buffer according to one of the methods on page 4. When processing small amounts of cells (.5000 cells). Add 3ul of linear acrylamide and 1ul Carrier RNA to the lysate before homogenization.

        4. Centrifuge at 13,000 x g for 3 minutes at room temperature when processing animal tissue.

        5. Transfer the supernatant to a new 1.5 ml centrifuge tube and add equal volume of 70% ethanol to the lysate. Mix thoroughly by vortexing or pipetting.

        6. Apply the mixture from step 5 onto HiBind® MicroElute® RNA column preinserted in a 2 mL collection tube. The maximum capacity of the spin cartridge is 750 ul. A precipitate may form on addition of ethanol in step 5. Vortex and add the entire mixture to the column. With the spin column inside a 2ml collection tube (supplied with kit), centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow-through and the collection tube.

        7. Place column in a new 2 ml collection tube (supplied), and add 400 ul RWC Wash Bufffer. Centrifuge and discard flow-through. Reuse the collection tube in step 8 or step 9. If on-membrane DNase I digestion is desired, proceed step 8, otherwise go to step 9.

        8. DNase I Digestion (Optional): this is point to start On-membrane DNase I digestion. (See detail procedure on page 14).

        9. Place column in the same 2ml collection tube and add 500 ul RWB Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and re-use the collection tube.

        Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for instructions

        10. Wash column with a second 500 ul of RWB Wash Buffer as in step 9. Centrifuge at 10,000 x g and discard flow-through. Then with the collection tube empty, centrifuge the column for 2 min at full speed ($13,000 x g) to completely dry the HiBind® matrix.

        11. Elution of RNA. Transfer the column to a clean 1.5 mL microfuge tube (not supplied with kit) and elute the RNA with 15-20 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Let it sit at room temperature for 2 minutes. Centrifuge 1 min at maximum speed.

        RNA may be eluted with a smaller (<15ul) volume of water to get higher RNA concentration. While reduced elution volume decrease total RNA yield. The total yield will be 20-30% less when the elution volume is less than 10ul.

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