The flashPAGE™ Fractionator is the fastest, easiest, and most reliable method for purifying mature miRNAs (19–23 nt) from longer precursor miRNA molecules, providing an efficient alternative to time-consuming standard polyacrylamide gel electrophoresis (PAGE) and subsequent gel elution. The procedure takes only 12 minutes and produces a small RNA fraction highly enriched in molecules <40 nt. Total RNA–previously isolated from a tissue or cell sample—is the typical starting sample for the flashPAGE Fractionator. Here one of our customers, Marc Bosse, Ph.D., of McMaster University, Hamilton, Ontario, shares a method he developed using whole cell lysates as the starting material for the flashPAGE Fractionator to purify endogenous mature miRNAs.

Streamlining the Protocol: Using Cell Lysates in Place of Total RNA

Dr. Bosse works with human embryonic stem cells (hESCs) grown in 6-well plates. He needed to profile the miRNAs in cells from a single well (2 × 106 cells) using microarray technology. In this case, the use of cell lysates in place of total RNA samples was crucial since he was working from small cell numbers and was challenged to obtain enough total RNA to provide miRNA sufficient for performing array analysis. The protocol he developed (see protocol in sidebar, Loading Cell Lysates Directly onto the flashPAGE Fractionator) not only resulted in better yields of the small RNA fraction, but also saved him substantial time and handling of the samples compared to experiments where he first isolated 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.

Results

Dr. Bosse compared the RNA recovered from the flashPAGE Fractionator when starting with total RNA versus his cell lysates. He found that he obtained a higher yield of enriched miRNA when he used cell lysates as the starting material, and the RNA has the same nucleic acid profile as that generated from a total RNA sample (Figure 1). Subsequent microarray analysis of these samples confirmed that the enriched miRNA sample from the lysates gave a similar profile to that derived from total RNA (data not shown).


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.


Scientific Contributor
Marc Bosse • MacMaster Univeristy, Hamilton, Ontario

PROTOCOL:

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).