TITLE: Purification of Total RNA from Cells Using Spin Technology
AUTHORS: Malu G Tansey

[DESCRIPTION]
Purification of Total RNA from Cells Using Spin Technology

[STEPS]
SECTION: Method
1. Add 20μl β-ME per 1 ml Buffer RLT. Dispense in a fume hood. Buffer RLT containing β-ME can be stored at room temperature for up to 1 month.
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2. Add volumes of ethanol (96–100%) as indicated on the bottle to concentrate buffer RPE to obtain a working Solution.   
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3.  Harvest cells (do not use more than 1 x 107 cells)  To lyse cells directly in the cell-culture vessel (up to 10am diameter): Determine the number of cells. Completely aspirate the cell-culture medium, and proceed immediately to step 4.   To trypsinize and collect cells (Cells grown in cell-culture flasks should always be trypsinized): Determine the number of cells. Detach cells from the dish or flask using trypsin, add medium containing serum to inactivate the trypsin, transfer the cells to an RNase-free centrifuge tube, and centrifuge at 1200rpm for 5 min. Completely aspirate the supernatant, and proceed to step 4.   
SECTION: Method
4. Disrupt the cells by adding Buffer RLT.  For direct lysis of cells: Add the appropriate volume of Buffer RLT as below to the cell-culture dish. Collect the lysate with cell scrapter. Pipet the lysate into a microcentrifuge tube. Vortex or pipet to mix, and ensure that no cell clumps are visible before proceeding to step 5.                                                                                                                                

 

For pelleted cells: Loosen the cell pellet thoroughly by flicking the tube. Add the appropriate volume of Buffer RLT as below. Pipet to mix, and proceed to step 5. 

 

SECTION: Method
5. Homogenize the lysate.  Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube,    and centrifuge for 2 min at full speed.   
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6.  Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting.
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7. Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube. Close the lid gently, and  centrifuge for 15 s at 10,000 rpm. Discard the flow-through  If the sample volume exceeds 700 μl, centrifuge successive aliquots in the same RNeasy spin column.  Discard the flow-through after each centrifugation.   
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8. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 10,000 rpm to wash the spin column membrane. Discard the flow-through
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9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 10,000 rpm to wash the spin column membrane. Discard the flow-through.   
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10. Repeat step7.   
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11. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 10,000 rpm to elute the RNA.    
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12. Always wear gloves and spray with RNaseZap and spread solution all over gloves
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13. Cell pellets can be stored at –70°C for later use or used directly in the procedure. Frozen cell pellets should be thawed slightly so that they can be dislodged by flicking the tube.
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14. Homogenized cell lysates from step 5 can be stored at –70°C for several months. Frozen lysates should be incubated at 37°C in a water bath until completely thawed and salts are dissolved. Avoid prolonged incubation, which may compromise RNA integrity. If any insoluble material is visible, centrifuge for 5 min at 3000–5000 x g. Transfer supernatant to a new RNase-free glass or polypropylene tube, and continue with step 6.   
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15. Perform all steps of the procedure at room temperature. During the procedure, work quickly.
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16. Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C