Working with RNAlater® (AM7021, Invitrogen)
Protocol for Cell Cultures (Adherent or Suspension)
Why use RNAlater?
RNA is impatient.
The moment you harvest cells, the clock starts ticking. RNases (enzymes that naturally exist inside cells and in the environment) begin breaking RNA down. If your samples aren't stabilized quickly, the RNA profile you worked so hard to capture can change before you even begin extraction.
RNAlater® acts like a "pause button." It rapidly penetrates cells and inactivates RNases, preserving the RNA expression profile at the moment of collection. That means you can harvest your cells today and extract high-quality RNA later, without worrying that the transcriptome has changed in the meantime.
Stabilizing Cell Cultures in RNAlater
Materials
RNAlater® Stabilization Solution (AM7021, Invitrogen)
Phosphate-Buffered Saline (PBS)
Centrifuge
RNase-free microcentrifuge tubes
Protocol
1. Pellet the cells
Harvest the cells by centrifugation using your laboratory's standard cell culture centrifugation conditions. Carefully decant or aspirate all culture medium.
2. Wash the cells
Resuspend the pellet in PBS to remove any remaining culture medium.
3. Pellet again
Centrifuge the cells once more and completely remove the PBS.
4. Resuspend the pellet
Loosen the pellet by gently tapping the tube. Resuspend the cells in the small residual volume of PBS that remains after aspiration.
If processing multiple samples, divide the suspension into 200 µL aliquots.
5. Stabilise the RNA
Add 5–10 volumes of RNAlater® directly to the cell suspension.
Example: Add 1.5 mL RNAlater® to 200 µL of cell suspension.
Mix thoroughly by gently pipetting up and down or inverting the tube.
Storage
Incubate the samples at 4°C overnight to allow RNAlater® to fully diffuse into the cells.
Transfer the samples to −20°C or −80°C for long-term storage.
Recovering Cells for RNA Extraction
Because RNAlater® contains a high concentration of salts and has a greater density than water, cells often do not pellet efficiently using routine low-speed centrifugation. Before RNA extraction, dilute the solution and centrifuge at a higher speed.
1. Dilute
Add an equal volume of cold PBS (1:1) directly to the stored sample.
2. Pellet the cells
Centrifuge at 3,000 × g to 5,000 × g.
Cells preserved in RNAlater® are more resistant to mechanical stress and tolerate this higher centrifugation speed without lysing prematurely.
3. Remove the supernatant
Carefully aspirate all of the RNAlater®/PBS mixture.
4. Proceed immediately
Add the appropriate lysis buffer from your RNA extraction kit and continue with the manufacturer's protocol.
Practical Notes
Remove as much culture medium and PBS as possible before adding RNAlater®.
Mix thoroughly after adding RNAlater® to ensure complete stabilization.
Always incubate the samples at 4°C overnight before freezing for long-term storage.
Low-speed centrifugation is often insufficient for recovering cells from RNAlater® because of its high density.
After dilution with PBS, centrifuge at 3,000–5,000 × g for efficient cell recovery.
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