Look forward to guaranteed yield available in nanomoles coming December 4, 2019!
The way oligonucleotides are quantified varies greatly between suppliers. The primary units of measurement are moles (e.g., nanomoles or nmol), molarity (e.g., micromolar or µM), and optical density at 260 nm (i.e., OD260). Some suppliers even include the “scale”, or number of reactions that you can perform with the quantity delivered.
This variation between measurements can be confusing. It makes it difficult to compare products across suppliers and makes it difficult to determine the quantity of oligonucleotides needed for a given experiment. Understanding how these different types of units are interrelated is key to ordering oligos and verifying your order. Here, we explain the units of measurement you may come across when ordering oligos and why IDT uses nanomoles in its guarantees for minimum oligo yield.
IDT typically delivers oligos in nanomole (nmol) or micromole (µmol) amounts. A mole is a unit of measurement, so a nanomole is 1 x 10–9 moles. A mole describes a group of molecules—specifically, 6.02 x 1023 of them. Avogadro’s number (6.02 x 1023) is used to describe very small things in a way that is similar to how we describe eggs or bakery items. One dozen eggs is equal to 12 eggs. One mole of a molecule is equal to 6.02 x 1023 molecules. The number of nanomoles of an oligo is unaffected by the weight, sequence, volume, or any other physical property of the oligo solution, which makes it a very straightforward measurement. Reporting the yield in nmol makes dilution calculations for your oligos simple and makes it easier to compare oligo concentrations.
IDT also provides information about the mass of the oligos. This value is most likely to be used when performing experiments in large scale. Mass can be conveyed in grams (g), milligrams (mg, 1 x 10–3 g), and micrograms (μg, 1 x 10–6 g). The molar mass, or how many moles are in 1 gram of the substance, can be used to find the total number of moles in the aliquot. From there, you can make the dilution calculation based on your desired final concentration.
The molar concentration considers the volume of the solution and is often measured in micromolarity (μM). Molarity is the number of molecules (or moles of molecules) per unit of volume (moles/liters). A micromolar solution describes the number of micromoles (1 x 10–6 moles) per liter. The concentration of reagents in qPCR protocols are often listed as μM or nM (nanomolar = 1 x 10–9 moles per liter). Oligos are provided either in solution or dried down. If it is dried down, you will determine the final molar concentration once you dissolve it in solution. If in solution already, the concentration will be reported. For example, if you order 100 nmol (0.1 μmol) probe in 100 μL (1 x 10–4 L), the concentration will be 1000 μM.
Optical density (OD or OD260)
Optical density (OD) is a common method for quantifying oligo concentration that uses the physical property of light scattering through a substance in solution. This characteristic is used to calculate the density of the solution using the Beer-Lambert Law, shown below. The Beer-Lambert Law describes the relationship between the concentration of a substance in solution and the amount of light scattered as it travels through the solution. The higher the concentration of oligos in the solution, the more light is scattered.
A = Ɛlc
A = absorbance
Ɛ = extinction coefficient
l = length of light
c = concentration in moles/liter
Absorbance is measured using a spectrophotometer. Variation can be introduced based on machine calibration. Proper mixing of the solution can also impact the final absorbance, since the same sample measured by the same machine might have a different absorbance if the substance is given time to settle.
The length of light refers to the width of the cuvette in which the sample is contained and loaded into the spectrophotometer. Standard cuvette width is 1 cm.
A problem with using OD to measure concentration is that the way each substance scatters light is unique to that substance. The unique properties of each substance are accounted for using a variable called the extinction coefficient (Ɛ), which you use to calculate concentration. Each oligo has its own Ɛ, and this value changes at each wavelength. Be sure to use the correct Ɛ for the wavelength used to measure absorbance. Even if 2 oligos are the same length, the specific sequence can also impact the Ɛ. 2 oligos with an OD of 1 can have drastically different concentrations.
Reaction number refers to the number of reactions that can be performed with a given solution. A supplier that reports reaction number assumes that you will only use their products for a specific application, at the recommended concentration. Reporting reaction number makes it difficult to further optimize primer-to-probe ratio or to use the supplier’s products in combination with products from another supplier. Reaction number can also be reported as a “fold concentration”. The reaction number can make it easier to calculate the quantity of a given product for your experiment, if your experiment is performed according to the supplier’s directions. Labs that run half reactions or that extensively customize their experiments may have difficulty calculating how much oligo is needed for a given reaction.
Understanding the way various suppliers report the amount of oligo and how much oligo is needed in your experiment will give you confidence when purchasing quality oligos at the best value. If you have any questions, please contact the IDT scientific application specialists at firstname.lastname@example.org.