RNA Isolation Made Easy
The PNA Story (Back to Top)
PNA is an analog of nucleic acids in which the phosphate backbone is replaced with a peptide-linkage. This modification, which was the brainchild of a group of Danes (ref. 1) confers a number of useful properties on the resulting molecule. The stability of PNA/nucleic acid triplexes (PNA can participate in Watson/Crick base pairing) is greater than DNA/DNA or DNA/RNA hybrids. PNA hybrids are resistant to attack by nucleases, and, more importantly for this discussion, PNA can invade the double helix, right through regions of secondary structure, to form stable triple helices.
Active Motif has developed a kit for isolating RNA using PNA, which promises to afford greater access to RNAs with extensive secondary structure that otherwise might be missed with standard oligo-dT isolation. In addition, this kit facilitates the isolation of the few percent of mRNAs that have poly-A tails too small to be captured by oligo-dT.
Active Motif's mVADER Kit, as it's called, uses a PNA analog, a phophono-PNA (pPNA), designed by Efimov (ref. 2) to get around some solubility problems encountered with classic PNA. The negative charge on the pPNA backbone, introduced with the phosphono moiety, improves PNAs solubility while not compromising its hybridization properties.
In the mVADER kit, pPNA is supplied in two forms: a linear chain of thymine pPNAs that hybridizes with open poly A tails of mRNA molecules, and what is called a clamping PNA molecule, which has two strands of 12-13 thymine pPNA's linked together (see diagram). In this form, PNA invades regions of secondary structure that exist around the poly A tails. Both molecules are tagged with biotin, which is then used to capture the hybrids with streptavidin-coated beads (supplied with the kit).
The resulting hybrids attached to beads can be released by incubating in water at room temperature. pPNA/DNA triplexes are more stable than pPNA/RNA triplexes, so through this elution step, mRNA, which is released from the hybrid, can be isolated away from DNA that remains bound. As an optional step to completely eliminate genomic DNA contamination, the researcher may do a DNAse treatment without worry since pPNA is resistant to nuclease digestion.
The proof is in the pudding
ActiveMotif scientists have used pPNA technology to isolate RNA transcripts in various forms, and have compared the results with those obtained with oligo-dT. The graph below shows some results obtained with the poly-T clamping pPNA and standard oligo-dT with single stranded RNA, double stranded RNA and RNA with a step loop structure. In all cases, the recovery is greater with PNA.
References
- P.E. Nielsen et al., "Sequence selective recognition of DNA by strand displacement with a thymine substituted polyamide," Science 254:1497-1500, 1991.
- Efimov et al., "Synthesis of poly-acrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications," Nucleic Acids Research 27:4416-26, 1999.
For more information: Active Motif, 5431-C Avenida Encinas, Carlsbad, CA 92008. Tel: 877-222-9543. Fax: 760-431-1351.
RNAture (Back to Top)
RNAture, a wholly own subsidiary of Hitachi Chemical Co., was spawned by an association between Hitachi and the University of California at Irvine that has been on-going for the last 10 years. Drawing on Hitachi's expertise in polymer chemistry, the company is focused on providing new tools for genomic research. The first to hit the streets is its mRNA Express Kit.
The technology used in the mRNA Express Kit combines a proprietary immobilization process on microwell plates, vacuum/pressure separation, and a novel contamination barrier that eliminates well-to-well crosstalk. The technology captures cellular poly A mRNA directly in the wells of thermocycler-compatible plastic microplates included in the kit through a three step process. First, cells are transferred by centrifugation, vacuum or positive pressure onto the membrane of the RiboCap 96-well filter microplate, which features an exclusive contamination barrier that prevents well to well crosstalk. Then the RiboCap is placed on top of the GenePlate, a 96-well microplate container with immobilized oligo-dT, and lysis buffer is added. Finally, the mRNA is transferred by spinning or applying vacuum or pressure onto the GenePlate, and incubated for sufficient time at room temperature to allow for capture of the mRNA by hybridization.
At this point the mRNA can be eluted or used to synthesize and amplify cDNA, using RT-PCR, on the GenePlate. The microplate functions as the solid support for quantitative reverse transcriptase-polymerase chain reaction and other applications, allowing researchers to move directly to the amplification step.
The mRNA Express Kit is compatible with fully automated operation by industry-standard liquid handlers and robotics systems. RNAture has in the works an enzyme pack and quantitative RT-PCR for selected genes. The company is projecting an aggressive product development pipeline for functional genomics and other aspects of gene analysis. While the initial products will be kit-based, future efforts will be directed toward DNA/RNA chips.
Applications
Click here to here to view some results obtained using RNAture's mRNA Express Kit, prepared by Shiliang Qin and Michael Brush, R&D scientists at RNAture.
For more information: RNAture Inc., 1003 Health Sciences Rd. West, Irvine, CA 92612-3054. Tel: 877 327-8762. Fax: 949-725-2788: e-mail: info@RNAture.com.
By Laura DeFrancesco