Hints and Tips

Handling of DNA Oligos

When handling DNA oligonucleotides, please consider the following:

• Make sure you use nuclease-free solutions only (see our suggestion below for potential solvents)
• Handle the DNA oligonucleotides carefully to avoid bacterial contamination
• Oligos are generally speaking more stable at higher concentrations
• Some fluorescent dyes (in particular Cy dyes) are especially light sensitive, and exposure to light should be minimized. Furthermore, some dyes may be sensitive to oxidation. Therefore, please ensure that you only open the tube if required.

How Do You Dissolve Your Oligo?

Generally speaking, oligonucleotides are best dissolved in sterile water or 10 mM Tris-HCl buffer (pH 7.5). However, there are a couple of exceptions that require special attention:

• Oligos carrying a fluorescent dye should not be dissolved in distilled water. This is because distilled water usually has a pH of ~6.0 which favors the degradation of the fluorescent dye. In such cases, you should only use 10 mM Tris-HCl buffer at pH 7.5.
• Oligos carrying fluorescent dyes of the cyanine family, such as Cy3 etc. should not be dissolved in 10 mM Tris-HCl buffer at pH 7.5. This is because they degrade slowly at pH >7.5. In such cases, you should only use distilled water.

How Do You Re-suspend Your Oligo?

• Do a short spin at max. speed in a centrifuge to collect the pellet at the bottom of the tube
• Add an appropriate amount of sterile water or buffer
• Heat the tube for 5 minutes at 65°C
• Vortex or mix by pipetting vigorously up and down

How to Anneal Complementary Oligos?

Option 1:
Materials:
1x annealing buffer (i.e. Tris-HCl 10 mM, pH 7.5-8, 20 mM NaCl or alternative commonly used buffer)

• Dissolve your Oligos at 200 µM in annealing buffer. Therefore, you should use one-half of the volume of the buffer given in the technical datasheet
• Mix the two Oligos in a 1:1 ratio
• Heat the solution to 92°C and keep for 2 min
• Cool to RT by removing the tube from the heating source

Your double strand will be annealed as 100 µM (100 nmol/ml) in Tris-HCl, pH 7.5-8, 20 mM NaCl (or alternative buffer)

Option 2:
Materials:
5x annealing buffer (i.e. Tris-HCl 50 mM, pH 7.5-8, 100 mM NaCl or alternative commonly used 5x buffer)

• Dissolve your Oligo at 100 µM in pure water (information on the volume given is provided in the technical datasheet). Alternatively, you can order the oligos already dissolved as 100 µM (100 nmol/ml) from Microsynth
• Mix the two oligos in a 1:1 ratio
• Add the buffer in a ratio of 4 parts oligo-solution and 1 part 5x annealing buffer 4:1 (v:v)
• Heat the solution to 92°C and keep it at that temperature for 2 min
• Cool to RT by removing the tube from the heating source

Your double strand will be annealed as 40 µM (40 nmol/ml) in Tris-HCl, pH 7.5-8, 20 mM NaCl (or alternative buffer)

Storage of DNA Oligos

If you want to store oligonucleotides, please consider the following:

• Please avoid repeated thawing and freezing as the physical forces involved may degrade your oligos
• Depending on your experiment, please choose a suitable storage condition (we refer you to our suggestions for potential storage conditions set out further below)
• Preparing aliquots may make sense if oligos must be stored and used over a long time period without losing activity

Handling of RNA Oligos

When handling RNA oligonucleotides, please consider the following:

• Make sure that you work RNase-free and that you use nuclease-free solutions only (see our suggestion below for potential solvents).
• Please handle carefully to avoid bacterial contamination.
• Oligos are generally speaking more stable at higher concentrations.
• Some fluorescent dyes (in particular Cy dyes) are especially sensitive to light and should be stored in light-tight tubes. Furthermore, some dyes may be sensitive to oxidation. Therefore, please ensure that you only open the tube(s) if required.

How Do You Dissolve Your siRNA or Single-stranded RNA?

Generally speaking, oligonucleotides are best dissolved in sterile RNase-free water or 10 mM Tris-HCl buffer (pH 7.5). However, there are a couple of exceptions that require special attention:

• Oligos carrying a fluorescent dye should not be dissolved in distilled water. This is because distilled water usually has a pH of ~6.0 which favors the degradation of the fluorescent dye. In such cases, you should only use 10 mM Tris-HCl buffer at pH 7.5.
• Oligos carrying fluorescent dyes of the cyanine family such as Cy3 etc. should not be dissolved in 10 mM Tris-HCl buffer at pH 7.5 since they degrade slowly at pH >7.5. In such cases, you should only use distilled water.

How Do You Re-suspend or Anneal Your siRNA?

siRNA is usually delivered as annealed, purified, and ready-to-use duplexes (40uM) in annealing buffer (10 mM TRIS-HCl pH 7.5 and 20 mM NaCl). However, on request, we deliver them in dried form as separate strands in two separate tubes. If you don’t know how to anneal, please observe the following protocol:

• Prepare 100 µM solutions of each RNA strand. Combine 30 µl of each RNA oligo solution and add 15 µl of 5x annealing buffer to reach a final volume of 75 µl. The final concentration of the duplex should be 40 µM.
• Incubate the solution for 1-2 minutes at 90-95 °C. From there, keep this solution at the work bench until it reaches room temperature (cooling should be relatively slow and take about 45-60 minutes). Centrifuge the tube briefly to collect all liquid at the bottom of the tube.
• Once annealed, duplex siRNA is much more resistant to nucleases than single-stranded RNA and is best stored at -20 ^°C. The 5x annealing buffer can be freeze-thawed up to 5 times.

How Do You Re-suspend Single-stranded RNA?

• Do a short spin at max. speed in a centrifuge to collect the pellet at the bottom of the tube
• Add an appropriate amount of sterile RNase-free water or buffer
• Heat the tube for 2-3 minutes at 90 °C
• Vortex or mix by pipetting vigorously up and down

Precautions against RNase Contamination

RNA is prone to degradation due to its free 2'-hydroxy-group. The main causes for degradation are the activity of RNases as well as the prolonged incubation in alkaline solutions. Please observe the following precautions to help keep your RNA intact:

• Always work with fresh, disposable plastic consumables (e.g. mark a bag of tubes and put it aside. Never grab into the bag, but instead, drop the tubes out of the bag. This also applies to Pipetman tips). If you must use glassware, please be aware that RNases can survive autoclaving – in this case, you should bake your glassware at 250 °C for at least 4 hours.
• Always wear gloves (RNases are ubiquitous). You should change gloves frequently (Please be aware that any surface you might touch with your gloves may have already been touched by a person who has not been wearing gloves.).
• Use RNase-free water/buffers. Most commercially available water is in fact RNase-free. Alternatively, you may produce DEPC-water. DEPC (Diethylpyrocarbonate) reacts with the primary amine groups, and therefore, inactivates RNases but cannot, for example, be used for Tris-buffers.

Storage of RNA Oligos

If you want to store oligonucleotides, please consider the following:

• Please avoid repeated thawing and freezing as the physical forces involved may degrade your oligos.
• Depending on your experiment, please choose a suitable storage condition (please refer to our suggestions for potential storage conditions further below).
• Preparing aliquots may make sense if oligos must be stored and used over a long time period without losing activity.

Design Guidelines for TaqMan Probes
Design Guidelines for Primers
General LNA Oligonucleotide Design Guidelines
Design Guidelines for siRNA's

Design Guidelines for TaqMan Probes

• Optimal length: 20 bases.
• Length range: 18-30 bases (a length of over 30 bases is possible; in this case, it is recommended that you use double-quenched probes and position an additional internal quencher at a distance of 8-10 bases from the fluorophore.
• Melting temperature (Tm): 68-70°C. Please ensure that the Tm of your probe is 8-10°C higher than the Tm of your primers (8°C for genotyping, 10°C for expression profiling).
• GC content: 30-80%.
• Avoid runs of an identical nucleotide, especially of 4 or more Gs (otherwise the interaction between the nucleotides can cause the oligo to form a secondary structure).
• Select the strand that gives the probe more Cs than Gs (due to the strong interaction that G displays with itself).
• Do not put Gs on the 5’end (quenches the fluorophore).
• When using within multiplex assays (allelic discrimination) consider the following:
  • Position the polymorphism in the center of the probe
  • Adjust the probe length so that both probes have the same Tm
  • If very short probes are needed (less than 18 bases), please get in touch with us

Design Guidelines for Primers

• Length range: 18-30 bases
• Melting temperature (Tm): 58-60°C
• GC content: 30-80%
• Avoid runs of identical nucleotides, especially of 3 or more Gs or Cs at the 3’end
• The total number of Gs and Cs in the last five nucleotides at the 3’end of the primer should not exceed two
• Primers should scan exon-exon junction. Contaminating genomic DNA will not be amplified by these primers

General LNA Oligonucleotide Design Guidelines

In contrast to standard DNA or RNA oligonucleotides, oligonucleotides with LNA nucleotides tend to be shorter due to their significantly higher melting temperature and the associated higher sensitivity. When designing LNA-containing oligonucleotides, the following general rules should be taken into account:

• LNA’s should be introduced at the positions where specificity and discrimination are needed (e.g. 3’end in allele specific PCR and in the SNP position in allele specific hybridization probes).
• Avoid stretches of more than 4 LNA nucleotides. LNA hybridizes very tightly when several consecutive residues are substituted with LNA nucleotides.
• Avoid LNA self-complementarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
• The typical primer length of 18-mer should not contain more than 8 LNA nucleotides.
• Each LNA nucleotide increases the Tm by approximately 2-4oC.
• Do not use blocks of LNA near the 3’ end.
• Keep the GC content between 30-60 %.
• Avoid stretches of more than 3 G DNA or LNA nucleotides.
• The Tm of the primer pairs should be nearly equal.

For very specific or novel assay settings, design rules may have to be established empirically. However,  following the above recommendations will represent a good start.

Design Guidelines for siRNAs

Finding the optimal design strategy of siRNA is an area of active research. Recently, several studies have underlined the importance of various factors for an effective siRNA. Including these (empirically derived) criteria in the design of a siRNA increases the chance of creating a specific and highly effective siRNA in terms of knockdown.

Important criteria include the Reynolds Criteria [1, 2]:

• The GC content of the siRNA should be 30% - 52%.
• Try to place at least 3 A/Us at positions 5-19 of the sense strand.
• Make sure that there is a lack of internal repeats (Tm of secondary structure < 20°C).
• Try to place an A at position 19 of the sense strand.
• Try to place an A base at position 3 of the sense strand.
• Try to place a U base at position 10 of the sense strand.
• Try to place a base other than G or C at position 19 of the sense strand.
• Try to place a base other than G at position 13 of the sense strand.

Please note that not all these criteria have to be fulfilled, but generally speaking, the more the better.

Our online siRNA design tool sorts the output with a score number that reflects these criteria and helps you to design your siRNAs quickly, reliably, and conveniently. It represents a customized tool that combines published design criteria with Microsynth’s own valuable experience. To use our design tool, please first login to our webshop and choose siRNA > siRNA Design Tool.

1. Boese et al. (2005). Mechanistic Insights Aid Computational Short Interfering RNA Design. Methods in Enzymology. Vol 392. Pages: 73-96
2. Reynolds et al. (2004). Rational siRNA design for RNA interference, Nature Biotechnology. Vol 22. Pages: 326-330

Traditionally, FAM-TAMRA is one of the most frequently used pairs for TaqMan probes in which FAM acts as the fluorophore and TAMRA as the quencher. Other quenchers can also be used, especially dark quenchers or black hole quenchers (BHQs) that capture energy from an excited reporter molecule without subsequent emission of light, i.e. they do not fluoresce.

Probes made by using dark quenchers tend to be more sensitive in quantitative detection systems. This is primarily due to lower background fluorescence and a better signal-to-noise ratio than probes that contain fluorescent quenchers. Moreover, dark quenchers enable the use of a wider range of reporter dyes, which expands the options available for multiplexed or genotyping assays.

Some fluorophores can be quenched by more than one quencher. In any case the absorption spectrum of the quencher needs to have a good overlap with the emission spectrum of the fluorophore to achieve optimal quenching. Quenchers have a quenching capacity throughout their absorption spectrum, but the performance is best close to the absorption maximum.

For multiplex PCR we recommend the following 5’ fluorophore 3’ black hole quencher combinations (are suitable for most qPCR thermo cyclers using TaqMan probes):

• Channel 1: FAM-MGBQ530
• Channel 2: HEX/JOE/Yakima Yellow*- MGBQ530 (*equivalent to VIC)
• Channel 3: ROX**-BHQ-2 (**equivalent to Texas Red)/ATTO550***-BHQ-2 (***equivalent to NED)
• Channel 4: Cy5-BHQ-2
• Channel 5: Dy681-BHQ2

Recommended final concentrations for standard experiments:

• Primers: 0.9 pmol/µl (0.9 µM)
• Probe: 0.2 pmol/µl (0.2 µM)

Recommended real-time-PCR conditions (Tm Primer: 59 °C, Tm Probe: 69 °C):

• 30 sec 95 °C
• 30 sec 57 °C
• 30 sec 72 °C, 35 cycles