What is lentivirus titer?
Abstract. The titer of a lentivirus vector is often expressed in transducing units per milliliter. This is a functional titer that reflects the lentivirus’ ability to transduce a particular cell line under specific conditions. Transduction of other cell lines is likely to be different and will require optimization.
How do you test for lentivirus titer?
The most accurate method of lentiviral titration uses qPCR to measure the number of proviral copies that have integrated into a target cell’s genome. This approach can discriminate multiple integration events, reducing the possibility of underestimating the viral titer.
How does qPCR measure viral titer?
So if your qPCR reads (for example) 100 copies/ul, your eluate will contain 300 x 100 (30 000, 3E4) copies, and this is the same as the number of copies in your pellet, so the viral titer would be 3E4/5E6 per cell or 3E4/1500 per ul.
How are titer values calculated?
For example, if the plate you selected was the 10^-5 plate, you would multiply 1570 by 10^5 to get 157000000. This final number is your phage titer, and represents the number of viruses per ml of your original culture.
How do you calculate MOI for lentivirus?
To calculate the amount of virus needed after establishing MOI:
- (total number of cells per vessel or well) x (desired MOI) = total TU needed.
- (total TU needed) / (TU/mL functional titer) = total mL of lentiviral particles to add to each well.
How long does it take for lentivirus to express?
All lentiviral vectors present in the transduction mix need at least 5 hours to penetrate the cells of interest. Based on the experiment, the transduction can be left from 5 hours to an overnight incubation.
Do you need to snap freeze lentivirus?
Recombinant lentivirus stock is more stable at ultra-low temperature, that is why lentivirus stock has to be snap-frozen in liquid nitrogen or dry ice/ethanol bath, and kept at ultra-low temperature, usually -60 °C to -90 °C. Lentivirus stocks become useless after several freeze-thaw cycles.
How is lentivirus titer measured by qRT-PCR?
Lentivirus titration by qRT-PCR The Lenti-X qRT-PCR Titration Kit provides a fast and simple method for measuring lentivirus titer. The kit uses a quick RNA purification step before quantifying the number of lentiviral genome copies using real-time PCR.
How long does it take to titrate a lentiviral vector?
Whereas traditional titration methods require up to 10 days to complete, these titration kits require only 4 hours and work with all HIV-1-based lentiviral vectors. Using qRT-PCR dramatically shortens the time interval between viral harvest and target-cell infection, allowing you to perform both on the same day.
How are lentiviral vectors used in molecular research?
Universal real-time PCR-based assay for lentiviral titration Lentiviral vectors are efficient vehicles for stable gene transfer in both dividing and non-dividing cells. This feature among others makes lentiviral vectors a powerful tool in molecular research.
Are there any primers that are compatible with lentiviral vectors?
*The primers included in this kit are designed to amplify a conserved region of HIV-1 genome adjacent to the packaging signal. These primers are compatible with all lentiviral vectors sold by Takara Bio and most commonly used lentiviral vectors.