Isolate Mature MicroRNAs from Cell Lysates Without RNA Isolation
Streamlining the Protocol: Using Cell Lysates in Place of Total RNA
The cell lysates were generated by simply disrupting the cells in flashPAGE Loading Buffer and then loading the mixture directly onto the flashPAGE Fractionator.
Figure 1. Cell Lysates Provide Enriched Small RNA Comparable to That from Total RNA Samples. Total RNA or cell lysates (cells resuspended in flashPAGE™ Loading Buffer) samples were fractionated on the flashPAGE Fractionator, and the small RNA fraction was run on denaturing acrylamide gels (15% Urea-PAGE). The gels were stained with ethidium bromide to reveal the nucleic acid.
Marc Bosse • MacMaster Univeristy, Hamilton, Ontario
Loading Cell Lysates Directly onto the flashPAGE™ Fractionator
(Note: this protocol was developed by Dr. Bosse and has been validated at Applied Biosystems.)
1. Collect Cell Sample. Human embryonic stem cells (hESCs) from 1 well of a 6-well plate (2×106 cells; wet weight: 15–20 mg) grow in clusters, and thus were treated successively with collagenase IV (20 min) and cell dissociation buffer (10 min) to generate a single cell suspension. The cell suspension was then centrifuged to pellet the cells.
2. Prepare flashPAGE apparatus. flashPAGE Lower Running Buffer (250 µL) was added to the lower buffer chamber, a flashPAGE Precast Gel was inserted into the chamber, and 250 µL of flashPAGE Upper Running Buffer was added into the Pre-cast Gel Cartridge.
3. Create cell lysate. The cell pellet was resuspended with 100 µL of Loading Buffer and disrupted by pipetting until a homogenous lysate was obtained.
4. Perform electrophoresis. The entire cell lysate volume was then loaded onto the upper chamber of the flashPAGE Fractionator, which was run for approximately 12–15 min (80 volts) until the indicator dye appeared in the lower chamber buffer.
5. Recover enriched RNA. The lower chamber buffer (250 µL) was recovered, and the chamber was carefully rinsed with 250 µL nuclease-free water, which was combined with the recovered buffer, resulting in a 500 µL volume.
6. Precipitate enriched RNA*. 60 µL 3M sodium acetate was added to the 500 µL recovered in step 5 and mixed briefly. This volume (560 µL) was then divided into 2 polypropylene microcentrifuge tubes, and 1 mL of 100% ethanol was added to each tube. The solution was mixed and stored overnight at –25ºC. The small RNA was collected by centrifugation at 20,000 × g for 45 min at 4ºC. Note that the RNA pellet is usually not visible from this small number of cells.
7. Prepare RNA for downstream applications. Pellets were carefully washed twice with 1 mL 85% room temperature ethanol, and tubes were air-dried by inverting them at 37ºC. The pellets containing the small RNA fraction were resuspended with 50 µL nuclease-free water and vortexed vigorously.
* Ambion recommends that you ethanol precipitate your RNA (do not use the flashPAGE Reaction Clean-Up Kit with this protocol).