- Highest recovery of mRNA of any available kit
- High stringency for lowering residual rRNA content
- Single round of selection sufficient for most applications
- Available in convenient magnetic particle format
High quality mRNA selection requires both efficient removal of rRNA and specific recovery of poly(A) RNA. Yield estimates based on mass alone are not indicative of either the quality or quantity of mRNA recovered since contaminating rRNA can significantly contribute to the mass of recovered RNA. The Poly(A)Purist Kits provide a level of mRNA enrichment unmatched by other protocols.
A Major Improvement in mRNA Isolation
The rRNA contamination that plagues mRNA enrichment is not exclusively caused by non-specific adsorption to the oligo(dT) matrix, but also by binding to, and co-purifying with, messenger RNA (mRNA). Researchers at Ambion have developed hybridization reagents and a protocol to minimize this unwanted interaction while still promoting efficient oligo(dT) selection. This patent pending methodology has been incorporated into the new Poly(A)Purist and Poly(A)Purist MAG Kits. The result is an mRNA selection procedure that typically requires only a single round of selection yet yields RNA that is more highly enriched and devoid of rRNA.
Don't Sacrifice Quality for Quantity
The superior performance of Poly(A)Purist is clearly demonstrated by a comparison of the mRNA recovered using 4 commercial kits and the MicroPoly(A)Purist and Poly(A)Purist MAG Kits (see the second figure at right). Identical total RNA samples (100 µg each) were processed with each kit according to the manufacturers' instructions. After mRNA selection, an equal percentage of the recovered RNA was evaluated on a denaturing agarose gel and visualized by EtBr staining. The intensity of staining is, therefore, proportional to the mass of recovered RNA.
As illustrated in the second figure at right, a popular magnetic bead-based kit (W), cellulose-based kit (X), and a latex bead-based kit (Z) yielded high mass amounts of RNA, but these samples contained a high level of residual rRNA contamination. The mRNA selected using a second magnetic bead-based kit (Y) appears to be of high purity, but (as illustrated in the third figure at right, sample Y) a significant percentage of the mRNA target is lost. These observations were confirmed by Northern analysis of the gel in Figure 1a, using 32P-labeled probes for ß-actin, GAPDH, and cyclophilin. When compared to the results obtained with the Poly(A)Purist Kits, the three samples with high rRNA contamination show a substantial reduction in signal. The signal from the RNA isolated using supplier Y's high stringency selection also has a markedly reduced signal, confirming that this kit's improved specificity results in the loss of a significant percentage of mRNA content from the sample.
Two Formats, Same Great Performance
The Poly(A)Purist Kits are available in two formats, one that uses oligo(dT) cellulose-based selection: Poly(A)Purist and MicroPoly(A)Purist, and one that utilizes oligo(dT) magnetic bead-based purification: Poly(A)Purist MAG Kit. Both cellulose and magnetic selection formats of Poly(A)Purist produce the highest possible yield of exceptionally clean poly(A)RNA.
The Poly(A)Purist and MicroPoly(A)Purist Kits utilize batch binding of RNA to pre-measured aliquots of oligo(dT) cellulose to circumvent the problems of slow flow rates and clogged columns often experienced during conventional, gravity-driven chromatography. A spin cartridge is used in the wash and elution steps for speed and convenience. Further time savings are realized because a single round of selection with Poly(A)Purist Kits is comparable to two or three rounds of selection using conventional technology. This vastly decreases the processing time required to isolate highly pure poly(A) RNA.
The Poly(A)Purist Kit contains reagents for 6 isolations, each from up to 2 mg total RNA, while the MicroPoly(A)Purist Kit contains reagents for 20 isolations, each from 2-400 µg total RNA.
The magnetic bead-based selection employed in the Poly(A)Purist MAG Kit delivers the benefits of increased processing speed, ease of handling, and scalability. Using the same hybridization and wash system as the cellulose-based Poly(A)Purist Kits, but with advanced magnetic particle technology, the entire selection procedure can be completed in as little as 45 min (see schematic at bottom right). In addition the reactions are completely scalable, allowing efficient selection from samples smaller than 100 µg of total RNA to very large (1 mg) total RNA samples.
The Poly(A)Purist MAG Kit contains reagents for up to 80 isolations, each from 100 µg of total RNA, or for 8 large preps from as much as 1 mg of total RNA.
The increased purity of mRNA isolated using Poly(A)Purist and Poly(A)Purist MAG results in more sensitive Northern, RPA, and RT-PCR based experiments, better templates for aRNA amplification, and higher specific activity probes with greater specificity for microarray analysis (top figure at left). In practice, more complete selection translates to better sensitivity in any procedure in which highly enriched mRNA is required as template or target.
Looking for the automated version?
Poly(A)Purist MAG-96 Automated has been discontinued as a stocked product, but is still available as a custom product. Email your request to custom@ambion.com.
Download Biomek® and MultiPROBE® programs for this kit at our Automation Resource.
Trademarks, Patents, Licensing
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