Multiplicity of Infection (MOI)

To set up a transduction reaction some simple math calculations will need to be performed. To
accomplish this, a number of variables in the experiment will need to be defined including the cell number,
MOI, total number of viral particles needed per reaction, and the titer of virus being tested.

 Cell Number—The cell number is just the number of cells present in a well that will be
infected with virus. For consistency simply use the number of cells plated the day before
transduction. Thus, from the plating example above it would be 3000 cells.
 MOI — The MOI is the number of viral particles per cell that is desired during the
infection reaction. The optimal MOI may have been determined for a particular cell line
already and if so just utilize that number for all AdenoSilence viruses. If not, then a range
of MOIs (e.g. 50, 250, 500, 1000, and 2500) will need to be assayed in an attempt to
identify the optimal infection amount. The optimal MOI should display a high
transduction efficiency with low cellular toxicity. Most MOIs will fall in the range of
100–2500 but they can be higher or lower.
 Viral Particles Needed— The total number of viral particles needed per reaction is
determined by multiplying the MOI times the number of cells plated per well. For
example, if 3000 cells were plated and an MOI of 1000 was desired then the calculation
would be:

Titer—The amount of AdenoSilence virus added to a reaction to obtain the total viral particles
needed is derived from the actual titer of that virus. Each AdenoSilence virus has a unique viral
titer which is listed as Viral Particles per Unit (VPU) on the certificate of analysis (COA)
provided for each virus. The VPU is also the same as viral particles per milliliter. To calculate
the amount of virus to add to the reaction simply take the viral particles needed (as determined in
the step above) and divide it by the titer of the virus. For example, if the titer of the virus from
the COA was 1x109 VPU then a continuation of the calculation from above would give:

 For consistency the virus that is delivered to the cells is normally diluted with cell culture medium
prior to addition. It is recommended that a standard delivery volume of 50 µL be utilized. To
determine how much medium should be mixed with the virus simply subtract the volume of virus
needed (from the above step) from the total delivery volume. Thus:
  Finally, the volumes determined above for virus and medium are multiplied by the number of
reaction wells to be tested plus an overage factor is added to account for pipetting errors (1.1X
per reaction well). Hence, for a reaction being done in duplicate wells the dilution of the virus
would be as follows:

 After gently mixing the medium and virus, quick spin the sample, and then add 50 µL per well.
Gently rock the plate for 10 seconds to mix the sample. The final volume in a single well of a 96-
well plate would be 150 µL.
 The cells can then be incubated at the standard cell culture conditions until the desired harvest
time. Typically there is no reason to remove the virus-containing medium from the cells following
transduction. If viability is a concern the medium can be replaced with virus-free medium at any
point 6 hours after the addition of virus.
 The above calculations must be carried out for every virus being tested since the titer (or VPU) for
each is likely unique. Additionally, if the MOI or cell number changes then the calculations will
need to be reworked as well.

Calculate MOI

(Total # cells per well)x(Desired MOI) = Total transducing units needed (TU)

(Total TU needed)/(TU/ml reported on C of A) = Total ml of lentiviral particles to add to each

well

Example

200 µl viral stock containing 10^6 (1e6) lentiviral transducing particles (5e6 particles/ml)

MOI= 5.0

 70,000 cells/well x 5 MOI = 350,000 TU

 350,000 TU / 5e6 = 70 µl of viral stock

MOI= 0.5

 70,000 cells/well x 0.5 MOI = 35,000 TU

 35,000 TU / 5e6 = 7 µl of viral stock