With intense research in such areas as RNA interference (RNAi) and micro RNAs, there is renewed interest in studying and using RNA. DNA/RNA synthesizers are used to synthesize oligonucleotides for a variety of applications that include PCR, sequencing, microarrays, RNAi, antisense and others. Laboratories using RNA on a large scale are looking to synthesize their own oligonucleotides, while others are seeking reliable custom oligonucleotide providers. Reliability, scalability, flexibility, ease of use, throughput, cost efficiency and service are some of the features that people look for.
There are different types of RNA that can be synthesized for different applications—namely, unmodified RNA, modified RNA, RNA conjugates, RNA chimeras and labeled RNA probes. Unmodified RNAs are de-protected, de-salted, endotoxin- free products, while modified RNAs can have a variety of modifications, internally as well as at the 3’ and 5’ positions. Dual-labeled RNA probes have fluorescent quenchers and reporters tagged to the RNA molecule for use in applications such as PCR and microarrays, while RNA conjugates are coupled with cell-penetrating peptides for use in gene expression, antisense, cell delivery and uptake. There are several proprietary synthesis and de-protection technologies that are in use for synthesizing RNAs with high coupling efficiencies, fast de-protection and a high level of purity.
Mark Behlke, MD, PhD, chief scientific officer at Integrated DNA Technologies (IDT), deals with multiple scales of RNA synthesis on a routine basis. IDT uses all in-housedesigned- and-built synthesizers. “We have a column-based platform that handles all medium- to large-scale synthesis and a 96-well plate-based system that does mostly smallscale synthesis,” says Behlke. While the plate-based system handles small-scale synthesis around the 100 nmol range, columns are used for synthesis in the 250 nmol to micromole range and large-scale reaction vessels can be used for synthesis from 10 mg up to 10 g. Synthesizing oligos on a plate-based system is cheaper and more efficient than using the column-based system. However, it’s much more difficult to maintain high coupling efficiency in a plate-based system, because it’s an open architecture system, while a closed column is much easier to keep environmentally sealed from atmospheric water, which is detrimental to nucleic acid synthesis. “Our specially engineered plate-based system can be kept in an isolated and controlled environment that is friendly for oligo synthesis and maintains high coupling efficiency,” says Behlke.
Maintaining high coupling efficiency and product quality is also more challenging with RNA synthesis than it is with DNA synthesis. “Last year we were able to convert the platebased systems to do RNA synthesis,” says Behlke. Besides synthesis, purification and quality control are also important. IDT has set up an affinity-based purification system to be done in a rapid and cost-effective way on all its samples. “Our base price for siRNA duplexes and dicer substrates is now below $100 for full-catalog single orders, and obviously for large orders, the pricing can be substantially discounted,” says Behlke. “While we can still make HPLC-purified smalland large-scale RNAs like we always have, this approach offers a more economical, high-throughput alternative.”
Tanuja Koppal, PhD, is a freelance science writer and consultant based in Randolph, N.J.
Agilent
The eArray 5.4 online microarray design tool enables users to design custom microRNA (miRNA) microarrays. Study of miRNA is rapidly emerging as these abundant molecules are increasingly associated with some cancers, heart disease, and other disorders. Researchers can use proprietary pre-designed human, mouse or rat miRNA probes, or access miRNA sequences for all 87 species in Sanger 12.0 in the designs of their miRNA microarrays. Each array contains about 15,000 features and arrays are printed eight-per-slide, providing low cost per sample. When the design is complete, the file is uploaded to Agilent’s SurePrint fabrication platform.
www.agilent.com
Biomatrica
The RNAconcentrator is designed to concentrate dilute, aqueous RNA solutions from picogram amounts of starting material to only 10 μl. The unique stabilization medium allows for improved recovery as compared to traditional methods, as the thermo-stable properties of RNAconcentrator protect samples from further degradation. It is easy to use—samples are applied into the RNAconcentrator tube and dried down. The dried RNA sample can then be conveniently stored for up to 1 week at room temperature until ready for use. Concentrated RNA can be used directly in downstream applications without further purification, thus avoiding sample loss typically associated multiple wash steps.
www.biomatrica.com
Integrated DNA Technologies
The miRCat™ small RNA cloning system makes the creation of small RNA (including miRNAs, piRNAs and endogenous siRNAs) libraries from any primary RNA source time and cost efficient. The system permits cloning of very rare small RNAs and takes into account the natural variability in structure and sequence between species. It is compatible with most existing standard laboratory protocols for processes such as RNA extraction, purification and cloning. Based upon a pre-activated adenylated oligonucleotide linkering method, the system has been designed to make small RNA library creation easy for all researchers. It consists of three sequential protocols: RNA isolation and enrichment, followed by cloning linker attachment, and ending with an amplification and cloning phase.
www.idtdna.com
Life Technologies
The TaqMan® MicroRNA Megaplex products consist of three molecular tools designed to streamline the workflow for microRNA (miRNA) analysis, address the needs of researchers working with minute amounts of RNA, and provide broad, up-to-date coverage of known miRNAs expressed in biological samples from humans, mice, and rats. The Megaplex™ RT Primers consist of high complexity pools of novel stem-looped reverse transcriptase (RT) primers that reduce the number of RT reactions needed to profile miRNA expression. The optional Megaplex™ PreAmp Primers enable pre-amplification of starting RNA material when sensitivity is of the utmost importance. Incorporation of pre-amplification reduces the total RNA input needed to perform an analysis to as little as one nanogram.
www.lifetechnologies.com
