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E.Z.N.A.® Total RNA Midi Kit Protocol for Eukaryotic Cells and Tissues

互联网2013-11-13

859

实验原理

The E.Z.N.A. Total RNA Midiprep Kits use the reversible binding properties of HiBind^® matrix, a new silica-based material. This is combined with the speed of mini-column spin technology. A specifically formulated high salt buffer system allows more than 600 ug of RNA molecules greater than 200 bases to bind to the matrix. Cells or tissues are first lysed under denaturing conditions that practically inactivate RNases. After the homogenization process by either bead-milling or rotor-stator homogenizer, samples are then applied to the HiBind^® Midi spin columns to which total RNA binds, after few quick washing step, cellular debris and other contaminants are effectively washed away. High quality RNA is finally eluted in DEPC-treated sterile water

实验试剂

1. 2-mercaptoethanol

2. 70% ethanol in DEPC-treated sterile distilled water

实验设备

1. Swinging-bucket centrifuge capable of 5000 x g with adaptor for 15 ml tube.

2. Sterile RNase-free pipette tips and 15 centrifuge tubes

3. Disposable latex gloves

实验步骤

1. Disrupt cells or tissues with 2 ml of TRK Lysis Buffer. Remember to add 20 ul of 2-mercaptoethanol per 1 ml of TRK Lysis Buffer before use. Homogenize cells with a rotor-stator homogenizer or vortex.

2 ml of TRK Lysis Buffer is sufficient for 10⁸ cells or approximately 200 mg disrupted tissue (~3 mm cube). For difficult tissues, more than 10⁸ cells, or greater than 200 mg tissue, use 4 ml of TRK Lysis Buffer. However, use no more than 300 mg tissue.

For tissue culture cells grown in monolayer (fibroblasts, endothelial cells, etc.), lyse the cells directly in the culture vessel as follows. Aspirate culture medium completely and add TRK Lysis Buffer directly to the cells. Use 800 ul for each T35 flasks or 10 cm dishes, and 400 ul for each smaller vessels. Pipette buffer over entire surface of vessel to ensure complete lysis. Transfer lysate from all flask to a clean 15 or 50 ml centrifuge tube and proceed to step 2 below. (This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination.)

For cells grown in suspension cultures, pellet cells at no greater than 1,500 rpm (400 x g) for 5 min. Discard supernatant, add TRK Lysis Buffer, lyse by pipetting up and down, and transfer to a clean 15 or 50 ml microfuge tube. Proceed to step 2.

For tissue samples, homogenize using one of the methods discussed. Unless using liquid nitrogen, disrupt and homogenize samples directly in TRK Lysis Buffer/2-mercaptoethanol and proceed to step.

Note: incomplete homogenization of the sample will cause lower yields and clogging of the column. It is recommended to homogenize the sample with rotor-stator homogenizers since it normally produce better yield.

2. Spin at 5,000 x g for 10 min at room temperature. Transfer the supernatant into a new tube and add an equal volume (2ml or 4ml) 70% Ethanol to the lysate and mix thoroughly by vortexing.

3. Apply sample onto HiBind^® RNA Midi column. The maximum capacity of the Midi column is 3.5 ml. (Larger volumes can be loaded successively.) A precipitate may form on addition of ethanol in step 2. Vortex and add the entire mixture to the column. With the spin column inside the 15 ml collecting tube (supplied with kit), centrifuge at 4,000-5,000x g for 5 min at room temperature. Discard flow-through and proceed to step 4.

4. Wash column with RNA Wash Buffer I by pipetting 3.5 ml directly into the spin column. Centrifuge as above and discard the 15 ml collection tube.

Note: This the starting point if on-membrane Dnase I digestion (page 6) is desired.

5. Place column in a clean 15 ml collection tube, and add 3 ml RNA Wash Buffer II diluted with ethanol. Centrifuge at 4,000-5,000 x g for 5 minutes at room temperature. Discard flow-through and reuse the collection tube in step 6.

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

6. Wash column with a second 3 ml of RNA Wash Buffer II as in step 5. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 10 min at 5000 x g to completely dry the HiBind^® matrix.

7. Elution of RNA. Transfer the column to a clean 15 ml centrifuge tube (Not supplied) and elute the RNA with 250-500ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 5 min at 4,000-5,000 x g. A second elution may be necessary if the expected yield of RNA >500 ug.

Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 70°C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.

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