This prep uses Qiagen’s RNeasy Mini Prep Kit. If you have never used this kit before, please look through the full protocol here. The full protocol also has information about extracting tissues.
Before Starting
If using a bottle of RPE Buffer for the first time, add 4 volumes of ethanol, as indicated on the bottle
Disruption and Homogenization of Animal Tissue
Excise the tissue sample from the animal or remove it from storage.
- Remove RNAlater stabilized tissues from the reagent using forceps
Determine the amount of tissue. Do not use more than 30 mg.
Add 10 µL beta-mercaptoethanol to a conical tube for every 1 mL Buffer RLT Plus used.
Make 70% Ethanol.
Place the tissue into a tube for disruption and homogenization according to how the tissue was stored:
- For RNAlate stabilized tissues:
- If using the entire tissue, place it directly in to a suitably sized vessel for disruption and homogenization.
- If using only a portion of the tissue, cut it on a clean surface (clean weigh boat) with a clean razor and place it in to a suitably sized vessel for disruption and homogenization. The tissue is stable at ambient temperatures while cutting and weighing.
- For unstabilized fresh or frozen tissues:
- If using the entire tissue, place it directly in to a suitably sized vessel for disruption and homogenization.
- If using only a portion of the tissue, weight the portion to be used and place it in to a suitably sized vessel for disruption and homogenization. Do NOT allow the tissue to thaw.
- Remaining fresh tissue can be placed into RNAlater Reagent to stabilize RNA. However, previously frzen tissues thaw too slowly in the reagent, preventing the reagent from diffusing into the tissues quickly enough to prevent RNA degradation.
We use screw-top tubes with a single ceramic bead for homogenization.
Add the correct amount of Buffer RLT Plus to the vessel. (Use 600 µL Buffer RLT Plus for tissues stabilized in RNAlater Reagent or for difficult to lyse tissues.)
Amount of Starting Material | Volume of Buffer RLT Plus |
<20 mg | 350 µL or 600 µL |
20-30 mg | 600 µL |
Immediately disrupt and homogenize the lysate in Buffer RLT Plus using one of the following methods:
- TissueRuptor: Immediately disrupt and homogenize the tissue until it is uniformly homogenous (30 seconds). Proceed to Extraction and Clean-Up.
- Disruption with mortar and pestle followed by homogenization in a Qiashredder tube: Place the tissue in liquid nitrogen and grind thoroughly with a mortar and pestle. Decant tissue powder and liquid nitrogen into an RNase-free, liquid-nitrogen-collec, 2 mL microcentrifuge tube. Allow the liquid nitrogen to evaporate but do not allow the tissue to thaw. Add the appropriate volume of Buffer RLT Plus at this point and pipette the lysate directly into a QIAshredder spin column placed in a 2 mL collection tube. Centrifuge for 2 minutes at maximum speed. Proceed to Extraction and Clean-Up.
- Disruption using a mortar and pestle followed by homogenization using a needle and syringe: Place the tissue in liquid nitrogen and grind thoroughly with a mortar and pestle. Decant tissue powder and liquid nitrogen into an RNase-free, liquid-nitrogen-collec, 2 mL microcentrifuge tube. Allow the liquid nitrogen to evaporate but do not allow the tissue to thaw. Add the appropriate volume of Buffer RLT Plus at this point and homogenize by passing lysate at least 5 times through a 20-guage needle fitted to an RNase-free syringe. Proceed to Extraction and Clean-Up.
- TissueLyser II or TissueLyser LT: See TissueLyser Handbook or LissueLyser LP Handbook. Proceed to Extraction and Clean-Up.
Centrifuge the lysate for 3 minutes at maximum speed.
Carefully aspirate as much supernatant as possible. Do not disturb the ceramic bead.
If necessary, run the supernatant through a QIAshredder tube for 2 minutes at maximum speed.
Disruption and Homogenization of Animal Cells
Use a minimum of 100 cells. The recommended maximum is 3-4 x 10^6 cells. Read the original protocol for more information about loading specific cell types.
Do not overload the gDNA Eliminator spin column, as this will lead to copurification of DNA with RNA. Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and purity.
Add 10 µL beta-mercaptoethanol to a conical tube for every 1 mL Buffer RLT Plus used.
Make 70% Ethanol.
Harvest cells according to the following options:
- Cells
- Cells grown in a monolayer (do not use more than 1 x 10^7 cells):
- To lyse cells directly:
- To trypsanize and collect cells:
Cell pellets can be stored at -70°C for later use or used directly in the procedure.
Disrupt the cells by adding Buffer RLT Plus and pipette up and down.
- For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add the appropriate volume of Buffer RLT Plus. Pipette to mix and proceed to homogenization.
Number of pelleted cells | Volume of Buffer RLT Plus |
<5 x 10^6 | 350 µL |
5 x 10^6 - 1 x 10^7 | 600 µL |
- For direct lysis of cells grown in a monolyer, add the appropriate volume of Buffer RLT Plus to the cell-culture dish and collect lysate with a rubber policeman. Pipette the lysate into a microcentrifuge tube. Pipette to mix and proceed to homogenization.
Dish diameter | Volume of Buffer RLT Plus |
<6 cm | 350 µL |
6-10 cm | 600 µL |
Homogenize the lysate according to the following options (if processing ≤ 1 x 10^5 cells, they can be homogenized by vortexing for 1 minute):
- Pipette the lysate directly into a QIAshredder spin column placed in a 2 mL collection tube. Centrifuge for 2 minutes at maximum speed (21,300 xG). Proceed to Extraction and Clean-Up.
- Homogenize the lysate for 30 seconds using the TissueRuptor. Proceed to Extraction and Clean-Up.
- Pass the lysate through a 20-guage needle fitted to an RNase-free syringe at least 5 times. Proceed to Extraction and Clean-Up.
Homogenized cell lysates can be stored at -70°C for several months.
Extraction and Clean-Up
Transfer the supernatant in the collection tube to a gDNA Eliminator spin column placed in a 2 mL collection tube.
Centrifuge for 30 seconds at ≥ 8000 xG (≥ 10,000 rpm). Discard the column and save the flow through.
- Make sure no liquid remains on the column membrane after centrifugation. If necessary, repeat the centrifugation until all liquid has passed through the membrane.
Add 1 volume (either 350 µL or 600 µL) of 70% Ethanol to the flow-through, and mix well by pipetting. Do NOT centrifuge.
- For maximum RNA yields from liver, use 50% ethanol instead of 70% ethanol.
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.
Centrifuge for 15 seconds at ≥ 8000 xG (≥ 10,000 rpm). Discard the flow through and reuse the collection tube.
If the sample volume exceeds 700 µL, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow through after each centrifugation, and reuse the collection tube.
Add 700 µL Buffer RW1 to the RNeasy spin column. Close the lid gently.
Centrifuge for 15 seconds at ≥ 8000 xG (≥ 10,000 rpm). Discard the flow through and reuse the collection tube.
Add 500 µL Buffer RPE (with ethanol added) to the RNeasy spin column. Close the lid gently.
Centrifuge for 15 seconds at ≥ 8000 xG (≥ 10,000 rpm). Discard the flow through and reuse the collection tube.
Add 500 µL Buffer RPE (with ethanol added) to the RNeasy spin column again. Close the lid gently.
Centrifuge for 2 minutes at ≥ 8000 xG (≥ 10,000 rpm). Discard the flow through and reuse the collection tube.
Place the RNeasy spin column in a new 2 mL collection tube and discard the old collection tube.
Centrifuge for 1 minute at full speed (21,300 xG).
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.
To maximize yield, let the column sit for 1 minute before centrifuging.
Centrifuge for 1 minute at ≥ 8000 xG (≥ 10,000 rpm) to elute the RNA.
If the expected RNA yield is >30 µg, repeat the elution step using another 30-50 µL of RNase-free water, or using the eluate from the previous step. Reuse the collection tube from step 11.