Harvesting RNA from Cells grown in monolayer

Loading the Column

1. Harvest cells grown in a monolayer (do not use more than 10⁷ cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypzinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should alays be trypsinized. Determine the number of cells and completely aspirate the cell-culture medium.
2. Disrupt cells by adding 350uL of buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350uL of Buffer RLT and vortex or pipet to mix.
3. To homogienize the lysate: Pipet lysate directly into a QIA shredder spin column placed in a 2 mL collection tube, and centrifuge for 2 min at full speed.
4. Add 1 volume (350uL) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.
5. Transfer up to 700 uL of the sample, including anny precipitate that may have formed, to an RNeasy spin column placed in a 2ml collection tube. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. Discard the flow-through.

Washing the Column

1. Add 350uL Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. to wash the spin column membrane. Discard the flow-through.
2. Add the DNase I stock solution to 70 uL buffer RDD. Mix by gently inverting the tubem, and centrifuge briefly to collect residual liquid fro tmt the sides of the tube.
3. Add the DNase I incubation mix (80uL) directly to the RNeasy spin column embrane, and place on the benchtop (20-30^oC) for 15 min.
4. Add 350 uL buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at >8000 x g or >10,000 rpm. Discard the clow-through.

Elution

1. Add 500 uL buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at >8000 x g or >10,000 rpm. to wash the spin column membrane. This dries the spin colujmn membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
2. Place the RNeasy spin oclumn in a new 2 ml collection tube, and discared the flowthrough. Close the lid gently, and centrifuge at full speed for 1 min.
3. Place the RNeasy spin column in a new 1.5 ml collection rube. Add 30 uL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at >8000 x g or >10,000 rpm to elute the RNA.
4. If the expected RNA yield is >30 ug, repeat the previous step using another 30-50 uL RNase-free water, or using the eluate from the previous step. Reuse the collection tube from th eprevious step.