flashPAGE™ Fractionator System - Ultra-Fast Purification of Small Nucleic Acids

flashPAGE™ Fractionator System

The flashPAGE™ Fractionator System is the fastest, easiest, and most consistent method for PAGE purification of small nucleic acids. Unlike traditional Polyacrylamide Gel Electrophoresis (PAGE), which was designed for the visualization of small nucleic acids, the flashPAGE Fractionator System is designed and optimized for the purification of RNA and DNA.

The innovative flashPAGE Fractionator System enables:

Ultra-fast PAGE purification of nucleic acids—with standard gel run times of only ~12 minutes
Easy, streamlined protocol
- using ready-to-use reagents, just load your sample and run
- the "lower collection chamber" design eliminates the need for gel excision and elution
Consistent, reliable results with precise, ready-to-use gels and reagents

Consisting of the flashPAGE Fractionator apparatus, flashPAGE Pre-cast Gels, and flashPAGE Buffers (Figure 1), the flashPAGE Fractionator System provides an efficient alternative to laborious and time-consuming PAGE and subsequent gel elution, which have traditionally been required for small nucleic acid isolation.

Comparison of flashPAGE™ and Traditional PAGE Protocols.

Ultra Fast

The flashPAGE System is named to reflect the substantial time and labor savings over traditional PAGE purification of nucleic acids. With it, you will easily and efficiently purify small nucleic acid molecules in run times averaging only 12 minutes.

flashPAGE purification is based on passing up to 100 µg nucleic acids through a proprietary denaturing gel matrix, provided as pre-cast gel cartridges, into the lower buffer collection chamber. The lower buffer chamber has been designed for retention of eluted material to facilitate nucleic acid purification. In just a few simple steps, you'll eliminate hours to days of labor (see Figure 1).

Consistent Elution of Nucleic Acids

The flashPAGE Gels provide consistent separation of molecules, providing predictable separation of small, single-stranded nucleic acids within seconds. Using this system, you can purify RNA or DNA molecules of various sizes using time-based elution and standard controls.

The flashPAGE A40 Dye Marker, included in the flashPAGE Buffer Kit, elutes at the same molecular weight as a single-stranded RNA molecule of 40 bases. Under normal running conditions, this dye molecule elutes after approximately 12 minutes and can be used as a standard reference point for elution of nucleic acids.

With this system, you can rapidly purify nucleic acids smaller than 40 bases by terminating electrophoresis at the time of dye elution, visible through the instrument's front window. All molecules running through the gel are deposited in the specialized lower collection chamber and can be withdrawn at any point throughout the run. Figure 2 demonstrates the successful isolation of Ambion's Decade™ Markers (RNA molecules between 10–150 nt) using the flashPAGE Fractionator System. In this experiment, two samples were removed, separated by the flashPAGE A40 dye molecule. Following the PAGE run, the flashPAGE Reaction Clean-up Kit is available separately for rapid glass fiber filter (GFF) concentration of nucleic acids and removal of small molecular contaminants.

Figure 2. flashPAGE™ Fractionation. Decade™ Markers (Ambion) in a background of 10 µg mouse brain total RNA were loaded onto a pre-cast flashPAGE Gel Cartridge and electrophoresed with the flashPAGE Fractionator. Two successive fractions were collected. For the first fraction, the lower buffer was collected and precipitated when the dye indicator band had reached the lower end of the gel cartridge. After adding fresh lower buffer to the apparatus, the sample was electrophoresed for 10 more minutes. The second lower buffer fraction was then collected and precipitated.

High Performance

Each flashPAGE Pre-cast Gel can accommodate up to 100 µg total nucleic acid per column (approximately 10 ng of small RNA is recovered from a 100 µg total RNA sample). Extensive validation of the flashPAGE design confirms that greater than 95% of species longer than 40 nucleotides are excluded from the small RNA/DNA fraction when the run is terminated with the flashPAGE A40 Dye Marker. Faithful representation of RNA is maintained.

Advanced Instrumentation

Although the flashPAGE Fractionator was engineered with the most current design and mechanical circuitry, the apparatus is designed to be an easy addition to any lab. This instrument is provided with a power cord containing standard banana plugs at one end--thus permitting use with most common laboratory power supplies. The flashPAGE Unit to Unit Power Cable makes efficient use of gel power supplies by allowing up to four instruments to run in parallel from one power supply connection.

All components of the flashPAGE Fractionator System are manufactured under strict ISO 9001 requirements. Interested in learning more about this entire system?

Figure 3. Isolation of Mature microRNA Using flashPAGE™ Fractionator Procedure.

Scientific Contributors
Patricia Powers and Rick Conrad • Ambion, Inc.

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Using flashPAGE™ for miRNA Purification Research on microRNAs (miRNAs) is increasing as scientists begin to appreciate the broad role that these molecules play in the regulation of eukaryotic gene expression. Several hundred miRNAs have been identified in C. elegans, Drosophila, mouse, and humans, and their expression levels have been shown to vary between tissues and developmental states. Characterization of a number of miRNAs indicates that they influence a variety of processes, including early development [1], cell proliferation and cell death [2], and apoptosis and fat metabolism [3]. In addition, one study shows a strong correlation between reduced expression of two miRNAs and chronic lymphocytic leukemia, providing a possible link between miRNAs and cancer [4]. Although the field is still young, there is speculation that miRNAs could be as important as transcription factors in regulating gene expression in higher eukaryotes.

One of the challenges of miRNA analysis is purification of this fraction of small RNAs, while avoiding other contaminating small RNA species (e.g. tRNA, rRNA, precursor miRNAs, etc.). Since miRNAs are small and typically in low abundance, purification leads to increased sensitivity in miRNA analysis. In addition, accurate microarray profiling requires separation of mature miRNAs from the longer hairpin and pri-miRNA molecules (see Figure 3 at left).

Currently standard polyacrylamide gel electrophoresis (PAGE), followed by gel excision, and nucleic acid elution and concentration, are required for miRNA isolation and purification. This process is time consuming and often imprecise. The flashPAGE™ Fractionator System provides an alternative to PAGE and subsequent gel elution. Mature miRNAs are easily separated from precursor miRNAs and other small RNAs with the help of the flashPAGE™ A40 Dye Marker, which elutes at the same molecular weight as the mature miRNAs. The electrophoresis only takes 12 minutes, and there is no elution step.

References 1.   Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) Nature 403: 901–6.

2.   Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM (2003) Cell 113: 25–36.

3.   Xu P, Vernooy MG, Guo M, Hay BA (2003) Curr Biol 13: 790–5.

4.   Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM (2002) Proc Natl Acad Sci 99: 15524–9.