Description

The Retrovirus Titer Set is designed to quantitate viral RNA transcribed from retrovirus vectors based on the murine leukemia virus (MLV). The kit is based on real-time RT-PCR method to quantitate MLV RNA and accurately determine the virus titer. Use of this kit with the One Step SYBR PrimeScript™ RT-PCR Kit^* (Cat.# RR066A), provides a direct measurement of RNA titer from virus supernatant in around 4 hours. In addition, RNA titers of almost all MLV-based retrovirus vector can be measured because using the MLV packaging signal region as an amplification target.
The conventional method for measuring the infection titer of a retrovirus vector involves calculating the biological titer by infecting an indicator cell with the retrovirus and using fluorescence or drug resistance to measure infection efficiency. This method requires several days to complete, and cannot be used to measure a retroviral vector that lacks a marker or drug-resistance gene. In addition, when determined multiplicity of infection (MOI) based on biological titer, the procedure requires several days; thus, the virus supernatant must be kept frozen until infection. Freeze/thaw cycles can lower the virus titer and alter the MOI, and additional manipulations increase the likelihood of contamination.
This kit can measure retrovirus titer without marker or drug-resistance genes because it quantitates retroviral vector RNA. Because results can be obtained within several hours, infection at the obtained MOI is possible without freezing the virus supernatant.
The One Step SYBR^® PrimeScript™ RT-PCR Kit (Perfect Real Time), which is a one-step, real-time RT-PCR system including SYBR^® Green I, is recommended for use with the Retrovirus Titer Set. This kit combines PrimeScript™ reverse transcriptase, which has excellent fidelity and a rapid elongation rate, with Takara Ex Taq™ HS polymerase, a highly efficient, specific PCR enzyme.

* SYBR is a registered trademark of Molecular Probes, Inc.

Components

(for 100 reactions; 25 µl reaction system)^*1

Forward Titer Primer FRT-1 (10 pmol/µl)	50 µl
Reverse Titer primer RRT-1 (10 pmol/µl)	50 µl
RNA Control Template (5×108 copies/µl)*2	10 µl
EASY Dilution (for Real Time PCR)	1 ml×2
DNase I (RNase Free; 5 U/µl)	40 µl
10×DNase I Buffer	50 µl
RNase Inhibitor (porcine liver; 40 U/µl)	10 µl

*1 For Real Time RT-PCR (100 reactions) and DNase I treatment (20 reactions).
*2 RNA Control Template should be divided into small aliquots upon receipt to avoid multiple freeze/thaw cycles,
and stored at -70°C to -80°C .

Shipping

-20°C

Storage

Store at -20°C , except for the RNA Control Template, which should be stored in small aliquots at -70°C to -80°C.