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Total RNA Isolation From Laser dissected samples

互联网2013-11-13

851

实验原理

The E.Z.N.A.™ MicroElute® Total RNA Kits combine the reversible binding properties of HiBind® matrix, a new silica-based material with the speed of mini-column spin technology. A specifically formulated high salt buffer system allows 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 add the ethanol, samples are then applied to the HiBind® MicroElute ® columns to which total RNA binds, while cellular debris and other contaminants are effectively washed away. High quality RNA is finally eluted in DEPC-treated sterile water.

实验试剂

Regents supplied by user:

1. 2-mercaptoethanol (14.3M)

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

实验设备

Equipments supplied by user:

1. Sterile RNase-free pipette tips and microcentrifuge tubes

2. Disposable latex gloves

实验步骤

1. Collect the sample into a 1.5 centrifuge tube contains 65 ul or 300 ul TRK Lysis Buffer. Remember to add 20 ul of 2-mercaptoethanol per 1 mL of TRK Lysis Buffer before use.

2. Adjust the sample volume to 75 ul or 350 ul with TRK Lysis Buffer. When process small amount of cells (.5000 cells). Add 3 ul of linear acrylamide and 1 ul of Carrier RNA to the lysate before homogenization.

3. Mix the sample throughly by vortexing for 30 seconds to homogenize the sample.

4. Add 1 volume (75 ul or 350 ul ) of 70% ethanol to the homogenized lysate. Mix well by pipetting or vortexing.

5. Apply the mixture from step 4 onto MicroElute® RNA Column. The maximum capacity of the spin cartridge is 750 ul. A precipitate may form on addition of ethanol. Vortex and add the entire mixture to the column. With the spin column inside a 2ml collecting tube (supplied with kit), centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow-through and proceed to step 6.

6. Place column in a clean 2ml collection tube (supplied), and add 400 ul RWC Wash Buffer. Centrifuge as above and discard flow-through. Reuse the collection tube for next step.

Note: This the starting point if on-membrane Dnase I digestion (page 14) is desired.

7. Place column in the same 2 ml collection tube, add 500 ul RWB Wash Buffer diluted with ethanol. Centrifuge as above and discard the flow-through.

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

8. Wash column with a second 500 ul of RWB Wash Buffer as in step 7. Centrifuge and discard flow-through.

9. Then with the collection tube empty, centrifuge the empty column for 2 min at full speed ($13,000 x g) to completely dry the HiBind® matrix.

10. Elution of RNA. Transfer the column to a clean 1.5 mL centrifuge 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 center of membrane. Let it sit at room temperature for 2 minutes. Centrifuge for 1 min at full 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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