Retro-X qRT-PCR Titration Kit
Retro-X qRT-PCR Titration Kit
Brand: Takara Bio.
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Retro-X qRT-PCR Titration Kit
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 Ct values that are derived from a calibrated, serially diluted control viral RNA template.
Overview
- Measure retrovirus titers using TB Green-based, real-time PCR
- Retrovirus copy numbers are determined by comparing the sample's Ct to a standard curve derived from a calibrated control template
- Confirm the success of your retroviral packaging reaction to avoid wasting time with your experiments
- Perform consistent experiments, control copy number, and hence expression levels
- Ensure sufficient MOI so that 100% of your cells are transduced
- 200 qRT-PCR reactions* are included, and 10 different virus preps can be purified
*The primers included in this kit are designed to amplify a conserved region of the MMLV genome adjacent to the packaging signal. These primers are compatible with all MMLV vectors sold by Takara Bio (e.g., Q-series and L-series vectors) and most commonly used retroviral vectors. However, vectors carrying a significantly modified packaging signal, like the one carried by MSCV vectors, would be incompatible with the kit. If you are unsure if this kit is compatible with your vector, please send your vector sequence to Technical_Support@takarabio.com, and our support team will be happy to confirm primer compatibility.
Flowchart of the procedure used for titrating retroviral supernatants with the qRT-PCR titration kits
Flowchart of the procedure used for titrating retroviral supernatants with the qRT-PCR titration kits.
Expression increases with higher MOIs
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
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 109–104 copies 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 from Panel A demonstrates a strong linear correlation between Ct and the RNA copy number (log scale), with R2 = 1.000 and a PCR efficiency of 107.4%.
Lenti-X and Retro-X qRT-PCR titration kits offer the fastest titration methods
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.
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Flowchart of the procedure used for titrating retroviral supernatants with the qRT-PCR titration kits
Flowchart of the procedure used for titrating retroviral supernatants with the qRT-PCR titration kits.
Expression increases with higher MOIs
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
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 109–104 copies 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 from Panel A demonstrates a strong linear correlation between Ct and the RNA copy number (log scale), with R2 = 1.000 and a PCR efficiency of 107.4%.
Lenti-X and Retro-X qRT-PCR titration kits offer the fastest titration methods
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.
