‡ Phillips J., and Eberwine J.H. 1996. Antisense RNA Amplification: A Linear Amplification Method for Analyzing the mRNA Population from Single Living Cells. Methods 10: 283-288.
* All trademarks are property of their respective owners.
Beckman Coulter, the stylized logo, Agencourt, Biomek, RNAClean, SPRI, SPRIPlate and SPRIStand are registered trademarks of Beckman Coulter, Inc.
Agencourt Rnaclean
The Agencourt RNAClean system provides a simple, flexible, and highly reproducible process for purifying nucleic acid products generated in common enzymatic reactions. This method utilizes Solid Phase Reversible Immobilization (SPRI) paramagnetic bead-based technology. It is uniquely formatted for sample clean up of reverse transcription reactions (cDNA) as well as in vitro transcription (IVT) reactions (cRNA) in the Eberwine RNA amplification protocol‡ commonly performed prior to microarray analysis. This technique is easily performed in manual or automated formats and eliminates the need for vacuum filtration or centrifugation. The Agencourt RNAClean system delivers superior nucleic acid recovery and purity for use in downstream microarray gene expression experiments, and has been validated by Affymetrix* on their GeneChip* Array Station platform.
Application: Post-reaction cDNA and cRNA cleanup
Downstream Application Techniques: Gene expression via microarray analysis (using Eberwine RNA amplification protocol)
The Agencourt RNAClean XP system was developed to replace the current Agencourt RNAClean offering. As such, the
Agencourt RNAClean product line will be discontinued in 2010. Read the obsolescence letter.
Agencourt RNAClean Kit Features
- Purification of small and large nucleic acid products
- Complete removal of salts, unincorporated primers and dNTPs
- Simple 3-step protocol
- No centrifugation, filtration or precipitation steps required
- Elution in aqueous solution
- Purifies both cDNA and cRNA
Process Overview
1. Enzymatic reaction 2. Binding of cDNA to magnetic beads 3. Separation of total RNA bound to magnetic beads from contaminants 4. Washing of RNA with Ethanol 5. Elution of RNA from the magnetic particles
6. Transfer away from the beads into a new plate
6. Transfer away from the beads into a new plate
Effective Reaction Purification
The Agencourt RNAClean system quantitatively purifies a larger nucleic acid size range with a higher percent recovery than LiCl precipitation (Figure 1). In addition, the smaller fragments purified using Agencourt RNAClean maintain the same proportional representation as unpurified samples.
Efficient Nucleic Acid Recovery
Percent Recovery
| Agencourt RNAClean |
LiCl | |
| Total ladder (large) | 100 | 81 |
| 240 bp fragment | 100 | 89 |
| Total ladder (small) | 100 | 53 |
| 155 bp fragment | 100 | 40 |
Figure 1: Agencourt RNAClean kit vs. LiCl purification. Small (0.16-1.77 kb) and large (0.24-9.5 kb) RNA ladders from Invitrogen were purified using a standard LiCl precipitation protocol or Agencourt RNAClean kit. RNA ladders with and without purification were separated using gel electrophoresis (2% agarose gel - small ladder or 4% denaturing PAGE geo - large ladder). Samples were quantified using the Agilent* 2100 bioanalyzer and the percent recovery calculated based on unpurified ladder quantities.
Quality Purification for Quality Performance in Downstream Applications
Purity of the isolated nucleic acids is critical to their performance in downstream applications. The high quality of RNA purified using the Agencourt RNAClean system is shown in Figure 2. Quality of a 580bp in vitro transcript was analyzed using the Agilent 2100 bioanalyzer. A small reference peak and a single clean peak representing the purified transcript were observed. The Agencourt RNAClean kit is manufactured as an RNase-free reagent to eliminate RNA degradation during reaction purification. RNA purified from IVT reactions using the Agencourt RNAClean process was stable, showing no signs of degradation at room temperature for 2 days or for 2 hours at 37° C (Figure 2). Purified nucleic acids are also free of nucleases or other impurities that can inhibit downstream enzymatic reactions or direct transfection of transcribed RNAi (data not shown).
High Quality
Figure 2: Quality RNA purification. A 580 bp RNA transcript was purified using the Agencourt RNAClean system, incubated under various conditions and analyzed on a Agilent 2100 bioanalyzer for signs of degradation.
High Yield and Consistent Recovery
The Agencourt RNAClean system consistently recovers more cRNA than standard column-based clean up methods. Agencourt RNAClean is compared to RNeasy for cRNA clean up. When Agencourt RNAClean is used for both cDNA and cRNA purification, the recovery is higher with both the Affymetrix GeneChip One-Cycle Target Labeling kit and the Enzo the Bioarray High Yield RNA transcript labeling kit.
High Yield
Figure 3: Comparison of cRNA Purification Methods. Five micrograms of rat brain RNA were used in cRNA labeling kits from Affymetrix and Enzo. IVT incubation time was 8 hours. Key: cDNA clean up method/cRNA clean up method. (Testing done by Gene Logic, Inc.)
Ordering Information
To request a quote or make an inquiry use the on-line contact form.
In the United States and Canada call +1 800 361 7780.
Outside the United States and Canada consult the International Distributors list.
| Product | Size | Kit Components | Product # |
|---|---|---|---|
| Agencourt RNAClean Kit (60 mL) | 175 preps1 | Agencourt RNAClean Reagent | A29168 |
| Related Products | Product # |
|---|---|
| Agencourt SPRIPlate 96R - Ring Magnet Plate | A29164 |
| Agencourt SPRIStand - Magnetic 6-tube Stand | A29182 |
1 Based on 150 and 40 µL RT/IVT reaction volumes. One prep includes both the cDNA and cRNA cleanup.
