Updated : Tue, October 14, 2014 @ 10:21 PM
Originally published : Wed, Oct 15, 2014 @ 11:00 AM
Different oligonucleotide manufacturers deliver probes and primers in a variety of different measured units including moles (e.g. nmol), molarity (e.g. µM), optical density at 260 nm (e.g. OD), and even the number of reactions a scientist can perform. Although these measured units are all technically correct, it is important to understand what each unit is telling you so you know exactly what you are receiving. In this article, we will quickly cover the basics of each oligo delivery unit to help you make the most informed decision when placing your next order for probes and primers.
Quantity in Moles - abbreviated mol, commonly nanomole; (nmol)
This is the default delivery unit Biosearch uses for delivering oligos. The quantity of moles refers to the number of molecules present in the delivered amount. 5 nanomoles (nmol) of one probe will have the same number of molecules as 5 nanomoles (nmol) of another oligo even if the sequence and modifications are totally different. Because the quantity of moles is independent of the physical characteristics and sequence of each oligo compound it makes it a very convenient and transparent unit of measure. Similarly, the use of moles makes it very easy to dilute your oligos to a fixed molar concentration. When oligos are measured in moles, we know that we can compare one oligo to another very easily. This is not the case with units such as OD as we will see below.
Molar concentration - abbreviated M, commonly micromolar (μM)
The molar concentration (molarity) is always written with a big “M” such as in micromolar - μM. This unit refers to the number of oligo molecules per unit of volume. Almost every qPCR reaction protocol will refer to the molar (typically nanomolar, nM) concentration of oligo present in the reaction. If an oligo is sold in solution rather than dried down, then it will typically be delivered at a fixed concentration. For example, one might purchase 5 nanomoles (nmol) of probe delivered in solution at 100 micromolar (µM) concentration. The 5 nanomoles refers to the number of molecules which you purchased, and the 100 micromolar (µM) concentration simply refers to how concentrated those probes are within that liquid solution. Biosearch offers oligo delivery in solution to customers upon request.
Optical Density - abbreviated OD260 or simply OD
The OD at 260 nm is a physical characteristic of an oligo in solution. This refers to the amount of light scattered by the molecule in solution; literally how dense the substance appears to light. The OD260 is related to the concentration of an oligo in solution through the Beer-Lambert Law. Simply put, this law states that the amount of light scattered as a beam travels through a solution is directly related to the concentration of the oligo (or any substance) in the solution. So the more probe contained in the solution, the more light becomes scattered due to the higher concentration of oligo. Although this law is very useful for determining oligo concentration, this unit of measure becomes somewhat complicated by the fact that each unique oligo has its own unique light scattering properties. This is corrected for in the Beer-Lambert formula with a variable known as the extinction coefficient (EC260). Let us consider a simple example to demonstrate this difference using 1 OD of two simple oligos.
( OD260 / EC260 ) x 1,000,000* = Concentration in micromolar
*Conversion factor from moles to micromoles
|Oligo A||Oligo B|
|OD Measurement (260 nm)||1 OD||1 OD|
|Oligo concentration at 1 OD||7.31 µM||15.76 µM|
This is a somewhat of an unusual sequence example as these consist of only one base or the other. However, it conveys the concept that two oligos with identical OD values can have very different number of molecules. Since almost every qPCR protocol includes the oligos at fixed molar concentrations, the total number of reaction you could run would be limited by the lowest delivered oligo amount (oligo A). This highlights the need to understand your particular oligo’s characteristics.
Reaction number - commonly reaction number and fold concentration, such as 20X
Some manufacturers will presume that qPCR reaction conditions are fixed for all users, which prevent titration of oligo concentration to the optimal probe to primer ratio. In this case, the oligo maker is assuming you will use the probe or primer at their recommended concentrations and simply reports the amount of oligo as a fold concentration or is sold by number of possible reactions. This sort of quantity reporting is intended to simplify the ordering process and reaction setup. However, it is important to examine that your qPCR method aligns with the manufacturers. For instance, if you perform 10 microliter reactions and the manufacturer assumes you are running 20 microliter reactions you might purchase twice as many reactions than you actually need. Similarly, if you require a 250 nanomolar probe concentration in your reaction and the manufacturer assumes this to be only 100 nanomolar then you might end up with the ability to run only 40% of the reactions you originally intended. Knowing how much oligo you need for your research and how much is being delivered in your order is key to receiving the proper amount of material at the most economic prices.
We hope that this blog has shed some light on the different oligo delivery methods which exist in the market, and allows for users to feel more confident in their purchase of custom oligos. If you ever have a question about your oligo delivery options you can always contact our expert staff at firstname.lastname@example.org for advice.