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 SPRI® (Solid Phase Reversible Immobilization) 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 protocol1 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 Affymetrix2 on their GeneChip2 Array Station platform.

Application: Post-reaction cDNA and cRNA cleanup

Downstream Application Techniques: Gene expression via microarray analysis (using Eberwine RNA amplification protocol)


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
Format Flexibility
  • Tube-based
  • 96-well plate
  • 384-well plate
  • Fully automated
  • Manual


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

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.



  Agencourt RNAClean LiCl
 Total ladder (large) 100 81
 240bp fragment 100 89
 Total ladder (small) 100 53
 155bp fragment 100 40
Figure 1: Agencourt RNAClean kit vs. LiCl purification.
Small (0.16-1.77kb) and large (0.24-9.5kb) 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 Agilent2 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).


Figure 2: Quality RNA purification.
A 580bp 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.


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 hours3. Key: cDNA clean up method/cRNA clean up method.

References
1. 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.
2. All trademarks are property of their respective owners.
3. Testing done by Gene Logic, Inc.
* Testing done by Gene Logic, Inc.

Available for use in your laboratory
To Place an Order or Receive Technical Assistance: In the U.S. and Canada call us at 800-361-7780. Outside the U.S. and Canada call us at 978-867-2600. Email us at webinfo@agencourt.com.


Product Size Kit Components Product #
Agencourt RNAClean Kit (60 mL) 175 preps* Agencourt RNAClean Reagent 000494
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Related Products Product #
Agencourt SPRIPlate® 96R - Ring Magnet Plate 000219
Agencourt SPRIStand™ - Magnetic 6-tube Stand 001139
*Based on 150 & 40µL RT/IVT reaction volumes. One prep includes both the cDNA and cRNA cleanup.

Please contact your sales representative or distributor with any questions. In the U.S. and Canada, call us at 800-361-7780. Outside the U.S. and Canada, call us at 978-867-2600. Email us at webinfo@agencourt.com.