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^° 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.