Q: How does this kit work?
A: Our Retrovirus Quantitation Kit uses a polymer technology that pellets the virus from the media and uses a fluorescent dye to measure the retroviral RNA. The dye non-specifically binds to nucleic acid and is not virus-specific. However, any non-viral nucleic acids are separated from the virus during the assay protocol, so only viral RNA should be remaining at the detection step.
Q: Can this be used to titer retrovirus other than MMLV, such as MSCV?
A: The polymer technology used in this kit pellets the virus from the media based on size, rather than the nature of the envelope protein, so it will work with any retrovirus. The fluorescenct CyQuant® dye is used to measure the retroviral RNA, which non-specifically binds to nucleic acid and is not virus specific. This kit will work with any retrovirus, but the following points are critical:
1. The virus must be in cell culture media in order for the polymers to form a complex with the virus.
2. The sample titer must be above the kit detection limit, which is 1.5x10^9 VP/mL.
Q: Will this kit work with retrovirus made from pMXs vectors?
A: Yes, this kit is compatible with any MMLV-based retrovirus packaged using pMXs, pBABE, pWZL or similar retroviral construct.
Q: Can I concentrate the virus before using the kit?
A: Yes, here are the options for concentrating virus:
a. A concentrator unit with a 100,000 kDa molecular weight cut off membrane. Up to 2 mL of supernatant can be concentrated down to 200 µL, which should increase the concentration by 10-fold.
b. An ultracentrifugation step if the virus is VSV-G pseudotyped.
Q: What is the difference between physical titer and infectious titer?
A: Viral titers are represented in two ways: either functional/infectious titer in transduction units (TU/mL) or physical titer in viral particles (VP/mL). Physical titer is a measurement of how much virus is present and is usually calculated based on the level of viral nucleic acid or protein. The functional titer is a measurement of how much virus can infect the cell and is typically 100-1000 fold lower than physical titer. Functional titer is a more accurate measurement for calculating MOI but is sometimes not practical. Functional titer may be calculated from physical titer. Our Retrovirus Quantitation Kit measures physical titer.