Takara Retro-X qRT-PCR Titration Kit \200 Rxns

This Retro-X qRT-PCR Titration Kit and a real-time PCR instrument can be used to rapidly quantify the virion content of MMLV-based retroviral supernatants. The viral genome copy number in a supernatant sample is determined by first purifying the viral RNA, and then comparing the RT-PCR threshold cycle (Ct) of an appropriate dilution of purified viral RNA to sample curve Ctvalues that are derived from a calibrated, serially diluted control viral RNA template.

Lenti-X and Retro-X qRT-PCR kitration kits offer the fastest titration methods.Standard transduction-based titration methods require up to 10 days to complete.The Lenti-X and Retro-X qRT-PCR titration kits require only 4 hr, which enables virus harvest, titration, and infection of target cells to all be performed on the same day.

Expression increases with higher MOIs.Histogram plots of NIH 3T3 cells transduced with increasing amounts of supernatant containing an amphotropic retrovirus that expresses the ZsGreen1 fluorescent protein. Greater amounts of virus increase the number of infected cells expressing ZsGreen1.Panels A–C. MOIs of 0.3, 3, and 30, respectively, resulted in 8%, 50%, and 97% ZsGreen1-positive cells having a fluorescence intensity greater than 10 (M1 population).

The qRT-PCR retroviral titration kit has a wide dynamic range.Panel A. Amplification plots of a qRT-PCR titration of the Retro-X RNA Control Template from 109–104copies using the Retro-X qRT-PCR Titration Kit. The assay shows a dynamic range of at least six orders of magnitude with low No-Template-Control (NTC) background.Panel B. A standard curve of the titration fromPanel Ademonstrates a strong linear correlation between Ctand the RNA copy number (log scale), with R2= 1.000 and a PCR efficiency of 107.4%.