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In this *TransMission* Transfection 101, we bring you a brief primer on the ‘N to P ratio,’ which is a concept you may have encountered in the context of transfection.

Here, we explain:

- What is the N to P ratio?
- How to calculate the N to P ratio
- Significance of the N to P ratio

If you’ve ever dabbled in farming or gardening, you may have heard of ‘NPK’ or seen the ratio of nitrogen to phosphorus to potassium displayed on bags of soil. These elements makeup essential nutrients for plants, and their proportion in soils and fertilizers has an impact on the course of plant development. Drawing this analogy to the realm of transfection, the N to P ratio describes the stoichiometry between protonatable nitrogen (N) in a transfection reagent and anionic phosphate groups (P) in a nucleic acid.

A quick glance at the N to P ratio of a transfection mixture, abbreviated N:P or N/P, can convey information about the net charge of the transfection complexes within it, which can have an impact on the course of transfection. For example, particles in a transfection mixture with an N:P of 3:1 are likely more positively charged than in a mixture with an N:P of 1:1.

Of course, not all transfection reagents incorporate nitrogen, and not all protonatable nitrogen atoms are protonated (depending on the pH of the transfection mixture). Nonetheless, N:P is a useful metric for describing the composition of transfection mixtures and for conceptualizing the transfection process mechanistically. See ‘Significance of the N to P ratio’ below for a discussion of ways N:P is known to correlate with transfection outcomes. But first, let’s go over how to calculate N:P, and conversely, given a desired N:P, how to calculate the quantity of reagent and nucleic acid to mix.

The N:P for a given transfection mixture can be calculated using the simple formula below:

N:P = (mol N) / (mol P)

Where ‘mol N’ is the number of moles of protonatable nitrogen from the reagent in the transfection mixture, and ‘mol P’ is the number of moles of anionic phosphate from the nucleic acid in the transfection mixture.

The amount of ‘mol P’ can be approximated using the following relationship:

3×10^{-9} mol of P / 1 µg of nucleic acid

(You can derive this relationship for yourself, given that the average molecular weight of a nucleic acid is ~330 g/mol per base and that there is 1 mole of P per 1 mole of base.)

Ascertaining the amount of ‘mol N’ may be trickier and will require knowledge of the molecular weight and chemical structure of your transfection reagent. You will need to know:

- What is the molecular weight of your transfection reagent?

(µg / mol transfection reagent) - What is the number of moles of protonatable nitrogen per mole of transfection reagent?

(mol N / mol transfection reagent)

Equipped with this information, you can then approximate ‘mol N’ in a given transfection mixture:

‘mol N’ = (mass of transfection reagent) / (molecular weight of transfection reagent) × (mol N / mol transfection reagent)

Example

Let’s calculate N:P for a hypothetical transfection mixture containing 4 µg of DNA and 1.6 µg of the simple transfection reagent 25 kDa linear polyethyleneimine (PEI).

There are approximately 12×10^{-9} mol of P in the mixture (4 µg of DNA × 3×10^{-9} mol of P / 1 µg of nucleic acid = 12×10^{-9} mol of P).

Assuming the molecular weight of the transfection reagent is 2.5×10^{10} µg/mol and there are ~580 protonatable nitrogen atoms per molecule of the transfection reagent, there are approximately 37×10^{-9} mol of N (1.6 µg of PEI / 2.5×10^{10} µg/mol of PEI × 580 mol N / 1 mol of PEI = 37×10^{-9} mol of N).

Finally, the N:P ratio in this hypothetical transfection mixture can be calculated:

37×10^{-9} mol of N / 12×10^{-9} mol of P = 3:1

If confronted with the opposite task (i.e. given a target N:P, determine the quantity of reagent to mix with nucleic acid), you can use the same relationships discussed above. It may be helpful to start your calculations with the mass of nucleic acid required for your transfection mixture. For example, given a desired N:P of 1:1, if you are transfecting 10 µg of RNA (i.e. 30×10^{-9} mol of P), your transfection mixture will require 30×10^{-9} mol of N. The mass of transfection reagent required can then be calculated using your preexisting knowledge about the chemical structure and molecular weight of your transfection reagent.

Why do Transfection Experts concern themselves with N:P? While to use some transfection reagents, you need to ‘do the math’ to figure out how much reagent to mix with nucleic acid, this is never the case when transfecting with *Trans*IT® transfection reagents. Each *Trans*IT® transfection reagent formulation and protocol has been designed for ease of use. Though it may be beneficial to optimize the amount of *Trans*IT® transfection reagent volume to use per transfection–check out this blog post on optimizing transfection–you will never need to calculate N:P.

Though not needed in practice, N:P can be useful for discussing “how transfection works.” For instance, the hydrodynamic radius of transfection complexes is often inversely correlated to N:P. (In other words, nucleic acids tend to be more condensed in mixtures with higher N:P.) And, as discussed earlier, N:P is proportional to the net charge of transfection complexes, which may dictate their electrostatic interaction with charged cell surfaces. Importantly, the kinetics of aggregation, uptake and intracellular dissociation of complexes may also be correlated with N:P, depending on the reagent. These correlated properties of transfection complexes can ultimately impact transfection efficiency and cytotoxicity.

*Why* certain steps and parameters are suggested in transfection protocols can also be contextualized with the concept of N:P. Obviously, mixing too much or too little reagent with your nucleic acid would seriously affect N:P. How about the complex formation solution used in your transfection experiment? As mentioned above, the N in N:P refers to the *protonatable* moles of nitrogen from the transfection reagent in the transfection mixture. The protonate-ability of a reagent, and thus its ability to confer positive charge to and associate with nucleic acid, is sensitive to the pH of the complex formation solution. And, as a final example, optimal transfection complex formation times may vary among different types of transfection reagents which may function best at a different range of N:P.

Do you use N:P in your research? Let us know at techsupport@mirusbio.com!

*Trans*Mission**Feedback or questions?** We’d love to hear from you. Email techsupport@mirusbio.com or call us at 888.530.0801.