E.Z.N.A.?TotalRNAMidiKitProtocolforEukaryoticCellsandTissues

實驗概要

E.Z.N.A.^?  Total RNA Midiprep Kit provides a rapid and easy method for the  isolation of up to 600 ug of total RNA from cultured eukaryotic cells,  tissues, or bacteria. The kit allows single or multiple, simultaneous  processing of samples in less than 40 min. Normally, 5 x 10⁶ - 1 x 10⁸ eukaryotic cells, 5 x 10⁸-1 x 10¹⁰  bacterial cells, or 25- 200 mg tissue can be used in a single  experiment. There is no need for phenol/chloroform extractions, and  time-consuming steps such as CsCl gradient ultracentrifugation, and  precipitation with isopropanol or LiCl, are eliminated. While this kit  may be used for isolation of RNA from whole blood, we recommend you use  the E.Z.N.A.^? Blood RNA Kit (product # R6614/R6615) as it is  specifically designed for effective hemolysis and hemoglobin removal and  gives higher RNA yields.

RNA purified using the E.Z.N.A.^? Total RNA method is ready  for applications such as RT-PCR*, Northern blotting, poly A  RNA (mRNA)  purification, nuclease protection, and in vitro translation.

實驗原理

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

主要試劑

1. 2-mercaptoethanol

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

主要設備

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

實驗步驟

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.