Frequently asked questions

Oligonucleotides

Oligonucleotides

  • Spin the tube briefly to collect the pellet in the bottom of the tube.
  • Add an appropriate volume of sterile TE buffer (10 mM Tris-HCl, 0.1-1 mM EDTA ; pH 7.5-8.0) or dH2O.

We recommend preparing a stock solution at 100 µM concentration which can be achieved by adding a volume (µl) of sterile TE buffer or dH2O, equal to ten times the number of nanomoles of sample present in the tube.

For optimal long-term storage of fluorescent dye-labeled oligonucleotides, it is recommended that the oligonucleotides be resuspended in a slightly basic solution (i.e., sterile TE buffer at pH 8).
Exception: Cy® dye-labelled oligonucleotides should be resuspended at pH 7.

(si)RNA should be resuspended in RNase-free buffer to a convenient stock concentration (20 to 50 µM) and in small aliquots to avoid multiple freeze thaw cycles and contact with RNases.

  • Allow the tube to stand for a few minutes at room temperature, then vortex it for 15 seconds.

Please note that some oligonucleotides (i.e. milligram amounts or phosphorothioate oligonucleotides) are particularly difficult to resuspend and may require longer incubation times and/or thorough vortexing.

Heating may also help to speed up the process.

The synthesis scale refers to the amount of raw material used to start the synthesis of oligonucleotides.

The yield corresponds to the amount of final product recovered at the end of the synthesis and purification processes.

The oligonucleotides production yeld may be influenced by the following factors:

  • The presence of one or multiple modifications
  • The nature of the modifications
  • The coupling method and efficiency
  • The purification method

To quantify your Oligonucleotides, make an aliquot of the resuspended Oligonucleotides to a final volume of 1 ml of dH2O and vortex for a few seconds.

Measure the absorbance of this dilution at 260 nm (A260). Use the formula below to calculate the concentration of Oligonucleotides in your stock solution.

This formula is valid for an absorption of A260 ≤1.2.

Concentration in µg/ml = A260 x dilution factor x Weight per OD of stock solution (in µg/OD)

1 OD260 (Optical Density) unit is defined as the amount of oligonucleotide which, when dissolved in a volume of 1.0 ml results in an absorbance of 1.0 when measured at 260 nm in a 1 cm path-length quartz cuvette.

1 OD260 unit corresponds to approximately 33 μg of single strand DNA. These relationships, however, can be inaccurate for short fragments of DNA, such as Oligonucleotides. Base composition and even linear sequence will affect optical absorbance. Hence the precise value of the OD to mass relationship is unique for each oligonucleotide.

Example:

1.0 OD260 of CCCCCCCCCC (10 bases) equals 39 μg
whereas 1.0 OD260 of AAAAAAAAAA (10 bases) equals only 20 μg

The following equation shows the relation between the oligo amount in nanomoles and the OD 260 value


Nanomoles = (OD260/ε260)x106
ε260 is the extinction coefficient at 260 nm


Example:
1 OD260 unit of primer M13 Forward,
5’-GTA AAA CGA CGG CCA GTG-3’
Molar extinction coefficient (ε260) = 182.800 L / (mole x cm)
Nanomoles = (1.0 / 182.800 ) × 106 = 5.47 nmoles

Using the following equation, it is quite simple to calculate the amount in microgramme from the nanomole value and the Molecular weight of the oligonucleotide.


Micrograms = Molecular Weight × Nanomoles × 10-3

Example:

1 OD260 unit of primer M13 Forward,
5’-GTA AAA CGA CGG CCA GTG-3’
Molecular Weight = 5558.7
Micrograms = 5558.7 × 5.47 × 10-3 = 30.4 μg

Molar Extinction coefficient can be calculate by the following formula

 

ε260=2x (∑1(n-1)εNearest Neighbour) - ∑2(n-1)εindividual + ∑1nεModification

where ΣNearest Neighbour is the nearest neighbour constant for a pair of bases, ΣIndividual is the constant for an individual base, and n is the length of the oligonucleotide.

Anhydrous MW (g.mol-1) = ∑individual base MW + ∑individual Modification MW - 63.98 + 2.016

 

For DNA bases: MW dA = 313.21; MW dC = 289.18; MW dG= 329.21; MW dT = 304.20; MW dU= 290.17; MW dI = 314.19

For RNA bases: MW DNA counterpart + 16. When determining the weight of Uracil (rU) start with dU and not dT

For LNA bases: MW dA = 313.21; MW dC = 289.18; MW dG = 329.21; MW dT = 304.20; MW dU = 290.17; MW dI = 314.19

For 2’ O-Methyl bases: MW DNA counterpart + 30.03. When determining the weight of mU start with dU and not dT

For phosphorothioated bases: MW DNA counterpart + 16.06

We can assist you in the design of  your qPCR probes, siRNA and miRNA

Learn more

Our Quality Control department performs regularly stability studies on different type of oligonucleotides. Each oligo is stored under different conditions and results are analyzed to provide to our customers the most accurate information regarding the stability of their products.

Stability statement

Oligonucleotides are produced on nucleic acid synthesizers using optimized phosphoramidite chemistry and fully automatic oligo synthesis.


Our proprietary synthesis platforms provide computer-controlled oligo synthesis, cleavage, and deprotection in 4 different processing formats.

Even though oligos are synthesized with the highest achievable coupling efficiencies, we always perform an additional purification step on every oligo batch.

This purification step ensures that even the simplest primer will be suitable for most molecular biology applications, such as PCR, RT-PCR, sequencing, and hybridization studies.

 

During DNA synthesis, each nucleotide is coupled sequentially (from 3' to 5') to the growing chain according to the standard b-cyanoethyl chemical reactions. Each cycle consists of:

  • Deblocking: the first nucleotide, attached to the solid support is deprotected by removing the DMT-protecting group. This produces a free 5' hydroxyl group to react with the next nucleotide.
  • Coupling: the next nucleotide is added to the reaction and is covalently attached (i.e. coupled) to the previous nucleotide.
  • Capping: any of the first nucleotide that failed to react is capped so that it will no longer participate at any subsequent steps.
  • Oxidation: the bond between the first nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain.
  • Deblocking: the 5' DMT group is removed from the second nucleotide to prepare it for further cycles.
    At the end of the oligo synthesis, the crude product is cleaved from the solid support (CPG or polystyrene beads) and purified using various methods.

Synthesis process

All of our custom oligonucleotides are synthesized with a hydroxyl group on both the 3' and the 5' ends.

However, if requested, we can synthesize your oligo with a 5' and/or 3' phosphate.

Coupling efficiency in oligonucleotide synthesis is greater than 99 percent and it is possible to synthesize a 200 mer oligonucleotide by special methods developed in the Eurogentec laboratory. We have already succeeded in synthesizing a 220 mer oligo.

However, even though we can produce long oligonucleotides with excellent purity in terms of length, there is another factor to consider. The many chemical steps during each cycle of oligonucleotide synthesis have a small probability of causing damage to the bases (base modifications). Some of these modifications are mutagenic and will result in a product with incorrect coding properties. Such a strand could be isolated during cloning. The base modifications are difficult to remove by standard purification methods used for long oligos and so the best way to avoid them is to be less ambitious about the length of the oligonucleotides you use. Moreover, yield and quality are affected by base composition.

For most practical purposes it is better to synthesize two 100 mer oligonucleotides and ligate them together using a short complementary oligonucleotide template to hold the two long oligonucleotides together for the enzymic ligation step. If necessary one or both of the long oligonucleotides can be chemically phosphorylated.

Special precautions must be taken when synthesizing G-rich oligonucleotides but this is not a problem for us.

Equally, care must be taken when using them in a biological application as they tend to aggregate and form tetrameric structures. This leads to insolubility. Formation of such aggregate can also hinder the ability of the oligo to hybridize to its complementary strand or target sequence.

To avoid this, G-rich oligonucleotides should be dissolved in buffer, heated to just below boiling point then very slowly cooled down in the presence of the complementary strand or target sequence.

Moreover, to avoid long stretch of G, Inosine (a universal base) can be substituted for some of the "g" residues to disrupt the tetraplex.

If you encounter problems using a G-rich oligonucleotide please contact us for further advice.

The melting temperature (Tm value) of an oligonucleotide is dependent upon the length of the sequence, the G+C content and the type and concentrations of cation present, particularly sodium ion, Na+. We are using the following formulae to calculate the Tm:

Recommended for primers from 14 to 20 bases :
Tm1 (°C) = 2 * (A+T) + 4 * (G+C) (Wallace-Ikatura formula)

Recommended for primers > 20 bases :
Tm2 (°C) = 81.5 + 16.6 * log10[0.05] + 0.41 * (%G + %C) - 675 / N = (81.5 - 21.597098) + 0.41 * (%G + %C) - 675 / N

where N is the length of the oligo. The formula we use takes into account the salts concentration of the reaction, as PCR is typically performed in the presence of ~ 50 mM monovalent cations (0.05 in the above formula).
For degenerated oligos, the lowest (%G + %C) value must be used. For oligos containing Inosine, length = (Length of the oligo) - (Number of Inosine bases). Tm calculation is inaccurate for LNA, PNA and may be inaccurate for oligos containing certain modified bases.

A=Adenosine
B=[G,T,C]
C=Cytosine
D=[G,A,T]
G=Guanosine
H=[A,T,C]
K=[G,T]
M=[A,C]
N=[A,T,G,C]
R=[A,G]
S=[G,C]
T=Thymidine
V=[G,A,C]
W=[A,T]
Y=[C,T]

We carefully measure the OD (Optical Density) value for your custom oligonucleotide by measuring the absorption at 260 nm using an UV spectrometer. This information is provided on the oligonucleotide Technical Data Sheet as: the number of OD260 units, the number of nmoles and the number of µg. The amount of oligo expressed in nmoles and µg is calculated from the OD measurement.

When we supply oligonucleotides on a large scale as freeze dried solids we weigh them and also measure the O.D. value as a solution in 1.5ml of water. The O.D. value is used to calculate the number of micromoles and micrograms of DNA. We do not use the weight in mgs for these calculations for the following reason:

A freeze-dried sample of DNA is highly hydrated as water binds very tightly to the major and minor grooves of DNA duplexes and to the edges of the bases, sugars and phosphates of single-stranded nucleic acids. Therefore when the freeze-dried pellet is weighed, the total weight cound be up to 50% water. So why do we weigh the sample? The answer is to make sure that the weight roughly corresponds to the O.D. value. In general (very roughly), 20 O.D. units of mixed sequence DNA will weigh 1 mg. If the weight is much too high relative to the O.D. value we would know that something is wrong. The usual cause for a discrepancy occurs with very short oligonucleotides. They are quite difficult to desalt after HPLC and therefore a high weight relative to the O.D. reading is indicative of contamination with salt. In such a case we would repeat the gel-filtration step to remove traces of salt, re-weigh the sample and re-determine the O.D. value.

Quality control (QC) is an indissociable part of any oligonucleotides synthesis process. Eurogentec Oligonucleotides are made using only the highest quality equipment and reagents to guarantee excellent results. All reagents used for oligo synthesis come from reliable suppliers, and each lot is extensively QC checked prior to use.

All our synthesizers are fitted with on-line (real-time) trityl analysis to ensure that synthesis of each oligo meets our stringent quality requirements. All oligos are routinely analyzed by optical density (OD260) measurement.

According to the oligo specifications, we perform quality controls for free as reported in the table below.

Oligo Type

MS1 UHPLC
Custom Oligonucleotides Unmodified
Modified 2
UltraPureGold™
qPCR Probes Double Dye Probes
Molecular Beacons
MGB Probes
RNAi Oligonucleotides siRNA Duplexes
NGS Oligonucleotides
Complex Oligonucleotides 3
Calibration Oligos
Universal Primers

MS: Mass Spectrometry; UHPLC: Ultra Performance Liquid Chromatography.
1Always provided up to 60 bases long Oligonucleotides.
2Except for SePOP desalted oligonucleotides.
3Optional.
For technical reasons this general rule may be adapted to provide you with the most suitable accurate oligonucleotide.

 

If requested, we can perform specific QC

 Read More

At the end of each coupling cycle, the 5' end deprotecting group: the trityl also called DMT, is removed from the added nucleotide. The trityl quantity is then measured and plotted in a paragraph. Since the chemistry that all oligo manufacturers use does not achieve a 100% coupling, the coupling efficiency is used to predict the quality of the oligo. At Eurogentec we demand a very high coupling efficiency before an oligo will pass our QC.

HPLC purification produces higher yields of purified oligonucleotides than cartridge purification. It gives additional feedback which simple cartridge purification cannot do by providing our skilled technicians with a detailed chromatagram which reveals the purity of the oligonucleotide and indicates if there are any problems which were not picked up in the trityl analysis (synthesis quality analysis). Some impurities in modified oligonucleotides are not removed by cartridge purification (e.g. a common impurity in HEX-labelled oligonucleotides).

Oligonucleotides can be modified by direct incorporation during the synthesis or by post-synthesis labeling.

Direct incorporation
3’ modifications
Since automated oligonucleotide synthesis is realized from 3’ to 5’, these modifications are only possible if the corresponding solid support (CPG column) is available and if the modification is compatible with the chemistries used during the synthesis.

Typicalexamples are 3’-phosphate, 3’ Biotin, 3’ FAM, 3’ DDQ I, 3’ BHQ-1®…

5’ and internal modifications
Many modifications can be directly introduced at the 5’ end or at internal positions of the oligonucleotides using the phosphoramidites. However these modifications need to support the somewhat harsh cleavage-deprotection conditions including a strong basic pH.

Typical examples are 5’ Biotin, 5’ Phosphate, 5’ Cholesterol, 5’ FAM, 8-Oxo-dA, Biotin-dT, DABCYL-dT…

Post-synthesis incorporation
Post-synthesis modifications may influence the yield of the reaction. A lower yield may result from poly-modifications and/or strong secondary structures. Two major post-synthesis reactions are used to introduce sensitive dyes or compounds that do not exist as phosphoramidites.
In the first case the label is conjugated to an amino-modified oligonucleotide (3’, 5’ or on a dT) using its amino-reactive version (N-hydroxysuccinimide (NHS) ester in most cases).
The second possibility (originally also used for synthesis of molecular beacons) is the addition of a maleimide-modified label to a thiol-modified oligonucleotide.

Yes, we can synthesize oligonucleotides with mixed normal backbone / phosphorothioate groups or with mixed normal sugars / 2'-O-Methyl sugar and mixed phosphorothioate / 2'-O-Methyl sugars. Other combinations are also possible.

If the chemicals are commercially available, Eurogentec can modify your oligo with it. Please contact us

RNA synthesis is not as straightforward as DNA synthesis. However, we have synthesized RNA 70 mers for customers and these have been used successfully.

Addition of multiple thymidine residues at the 5'-end of a PCR primer will change the mobility of the PCR product. Large number of T residues can be added. An alternative is to use a non-coding hydrophilic monomer. The best choice is hexaethylene glycol (hexaethylene oxide). Multiple additions of this monomer can be carried out routinely.

Yes, apparently so. Provided that the PCR primer is not very long (up to 30 bases) and the PCR product is not long, unpurified oligonucleotides will be efficient PCR primers. However, it is difficult to determine the quantity/concentration of a crude PCR primer because it will contain impurities such as protecting groups and short failure sequences that affect the UV spectrum. Hence, if the concentration of the primer is determined by UV absorbance (O.D. 260) you will not have as much of the full length primer in solution as you think. For this reason Eurogentec does not provide crude primers. Even Eurogentec "unpurified" oligonucleotides (Genomic oligos) are partly purified at no extra cost by SePop (Selective Precipitation Optimized Process) desalting. this ensures that short failure sequences and the largest part of contaminants have been removed.

Standard desalted oligonucleotides (Genomic oligos) are normally synthesized on the day the order is received and sent out the next day. Modified oligonucleotides are synthesized and purified in a short timescale but the precise time depends upon the nature of the oligonucleotide.

For more information, check our delivery time table

The discovery of an efficient and easy way to knock out gene expression at the mRNA level has resembled the seek of the Holy Grail for more than 10 years. Unfortunately, the use of classical antisense techniques (i.e. using various chemically modified forms of oligonucleotides) has often resulted in insufficient or non-specific suppression of gene expression. It has recently been shown that the presence of short double-stranded RNA (siRNA for Small-Interfering RNA) induces RNA interference in most eukaryotic species, as well as in cell culture. RNA interference (RNAi) leads to the inhibition of protein expression via the sequence-specific, dsRNA-mediated destruction of target messenger RNA (mRNA).
It is believed that this discovery will have a tremendous impact on the study of molecular and cellular processes, and on gene function deciphering.

In vitro transcription yields RNA molecules that are equally suitable for RNAi studies. Nevertheless, these experiments require expert skills at each step: in vitro transcription, purification, quantification... As a consequence, we frequently meet scientists irritated by the poor quality results they get after these tedious steps, and who have shifted to our user-friendly siRNA.

We use bioinformatics tools to select siRNA oligos with characteristics perfectly matching the last experimental results in this field.

Several studies have shown that the most efficient siRNA contains a dTdT (or UU) overhang at their 3' ends. Using dTdT is recommended for optimal stability of the siRNA duplex, however, UU overhangs work equally well.

It is advisable to test two to four siRNA sequences per gene. Nevertheless, in some cases, this number may vary from one to ten depending on your applications and particular needs.

Despite its extreme efficiency, the selected siRNA might not work in your cell system. If so, it is advisable to check the following points:

  • If no knock-out of the target gene is observed, it may be useful to analyze whether the corresponding mRNA was effectively degraded upon addition of the siRNA. Two or three days after transfection, the total RNA is extracted and subjected to further analysis. RT/PCR appears to be the method of choice since it is faster and far more sensitive than Northern blotting.
  • Check for any sequencing error or polymorphism in your target gene. It has been shown that a single base mutation in the pairing region of the siRNA duplex is sufficient to abolish RNAi.
  • Check that your cell line can effectively express the target mRNA.

Although similar to DNA synthesis, the additional 2'-OH group of RNA introduces considerable complexity to the RNA synthesis and requires a different protecting group.

All RNA oligos at Eurogentec (including siRNA) are made using monomers containing a 2'-O-tertiary-Butyl-dimethylsylil (TBDMS) protecting group, assuring robust and reliable synthesis.

All oligos are made with the standard phosphoramidite solid-phase synthesis technology.

When you order one of our siRNA sets, you receive single-strand siRNAs in separate tubes, either lyophilized (PAGE purified) or desalted. In addition, Eurogentec sends aliquots of Annealing buffer 5X and RNase-free water along with each siRNA oligo set.
Such conditioning may seem bothering but it has several key advantages by allowing you:
- to use each single oligo as a negative control.
- to test various combinations of modified strands.
Finally, even when ordered pre-annealed, it is highly recommended to perform a heat/cool step after resuspension.

Every researcher would tell it: "The choice of the right controls makes the whole difference between a good and a bad experiment". This adage is particularly true for RNAi studies.


Therefore, to maximize your result interpretation, the following precautions should be taken when using siRNAs:


- Always test the sense and antisense single strands in separate experiments.
- Try to use a scramble siRNA duplex. This should have the same nucleotide composition as your siRNA but lack significant sequence homology to any other gene (including yours). Some researchers use other types of negative controls such as : introducing 4 to 5 mismatches in the original sequence, flipping the middle four bases, or even reversing the entire sequence. In all cases, it is crucial to check that the negative control sequences will not silence another gene.
- If possible, knock-down your gene with two independent siRNA duplexes to control the specificity of the silencing process.

Most readily transfectable cell lines are suitable for RNAi studies.

However, each new assay should be first optimized (i.e. by using a range of siRNA concentrations, by testing different (transfection reagent volume - siRNA amount) ratios ...) F

or a successful optimization, pay attention to the following points:
- Use only healthy cell cultures to assure good transfection reproducibility (i.e. don't use cells passaged more than 50 times).
- Try to avoid antibiotics during plating an up to 2 days after transfection.

RNA interference was first used in immortalized cell lines (such as the unavoidable Hela) whether adherent or in suspension. Recent studies indicate that even transfection resistant primary cell lines are suitable for RNAi research.

We typically obtain 80 to 95 % inhibition at the mRNA level after 24 hours transfection. Inhibition at the protein level is highly dependent on the protein half life.

Custom oligonucleotides shipped dried or in solution are stable for 1-2 weeks at room temperature when unopened.
For long-term storage please check our stability statement.

Stability statement

Custom oligonucleotides are shipped dried or in solution. They are stable for 1-2 weeks at room temperature when unopened.

For long-term storage please check our stability statement

Stability statement