FAQs RNA mid/large Scale oligos
The label on the oligo tube shows basic information like oligo name, oligo sequence including modifications, oligo and order ID, yield of oligonucleotide (OD260), and molecular weight.
In addition, you will receive a synthesis report containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, yield of oligonucleotide (OD260), Tm and molecular weight.
Oligos are made using a DNA synthesizer, which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.
|I||Deblocking||The first base, attached to the solid support via a chemical linker, is deprotected by removing the protecting group (trityl-group). This produces a free 5´ OH group to react with the next base.|
|II||Coupling||The next base is activated and couples to the 5’-OH-group of the last base of the chain.|
|III||Capping||Any of the first bases that failed to react are capped. These failed bases will play no further part in the synthesis cycle.|
|IV||Oxidation||The bond between the first base and the successfully coupled second base is oxidized to stabilize the growing chain.|
|V=I||Deblocking||The 5´ trityl-group is removed from the base which has been added.|
Each cycle of reactions results in the addition of a single DNA base. A chain of DNA bases can be built by repeating the synthesis cycles until the desired length is achieved.
Every DNA base (in terms of DNA synthesis chemistry, we are speaking of phosphoramidite monomers and amidites) added during DNA synthesis has a dimethoxy-trityl (trityl) protecting group attached to the 5´-hydroxyl position. This acid labile trityl-group is bound to the 5’-end of each support-bound monomer and protects the corresponding base from undergoing unwanted chemical reactions during the synthesis cycle. The trityl-group is removed in the first step of each synthesis cycle, immediately before a new base is added, until the elongation of the nucleotide chain is complete. The final trityl-group is removed before delivery (Unless otherwise requested).
DNA synthesis is a complicated process, which has improved significantly over the last years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses; however, it is inevitable that we will occasionally have to re-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.
Unless requested, oligos are synthesized without either 3´or 5´ phosphate. The 5´ and/or 3’-phosphate is available as a modification at additional charge.
There is a normal degree of variation in the appearance of the supplied dry oligonucleotide pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of unmodified and dye-labeled oligo pellets may vary from powdery to hyaloid. The color of unmodified oligo pellets may range from transparent over off-white and yellowish to tan. The pellets of labeled oligos are colored according to the dye attached.
Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.
Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as
- n-1/n-x oligos
- Incomplete Deprotection
- Acrylonitrile adducts
- High Salt Content Identification
Moreover, it is the fastest and most efficient way to identify potential product mix-ups.
We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.
MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).
ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.
|Comparison MALDI-ToF and ESI-ToF|
|< 60 nts||+||+|
|> 60 nts||-||++|
|Photosensitive Modified Oligos||-||+|
Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.
For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.
Synthesis scale refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. This is the same for all manufacturers of synthetic DNA using standard phosphoramidite chemistry. When a synthesis scale of 40 nmole is specified, approximately 40 nmoles of the first base are added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences; hence it is not possible to produce 40 nmoles of full-length product from a 40 nmole scale synthesis. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control. Final yield is the actual amount that we guarantee to deliver.
Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:
1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 5 nmol.
1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 2.5 nmol.
Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.
The best way to store RNA is as a dry pellet at -20°C to -80°C and to avoid frequent freeze-thaw cycles. If you want to store your RNA in solution re-suspend the delivered pellet in an RNAse-free solution buffered at pH 7.4 - 7.6 and store at -20°C or less. We recommend that RNAs are re-suspended at a convenient stock concentration and stored in small aliquots to avoid multiple freeze thaw cycles.
For modified oligonucleotides – especially for fluorescent dye labelled oligos – you should minimize the probe’s exposure to light because of its bleaching effect. Additionally, we recommend storing dye labelled oligos highly concentrated, rather than in working dilutions, if you don’t use them immediately. The higher the dilution factor the faster the fluorescent activity decreases/fluorescence fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, and dilute them just before usage. Click here to download our guidelines for handling RNA.
We recommend to dissolve single stranded RNAs in 1x TE buffer (10 mM TrisCl, pH7.5, 0.1 mM EDTA; prepared under RNase-free conditions). This buffers the pH and chelates metal ions, which may contribute to RNA degradation. RNase-free water is also acceptable.
metabion's RNA oligonucleotides are delivered deprotected and purified – ready to use. They are RNAse free, but as RNA is highly susceptible to degradation by exogenous RNAses introduced during handling, it is essential that you conduct all handling steps under sterile, RNAse free conditions. Never handle RNA without wearing gloves. RNAse free reagents, barrier pipette tips and tubes should be used!
For more information, please read FAQ: How should siRNA oligonucleotides be stored?