Retrovirus Quantitation Kit

Sample

viral supernatant and purified virus

Intended Use

The Retroviral Quantitation Kit provides an efficient system for rapid quantitation of retrovirus titer for both viral supernatant and purified virus.

Contents of Kit

1. Solution A: One tube, 200 μL.
2. Retrovirus Solution B1: One tube, 800 μL.
3. Retrovirus Solution B2: One tube, 800 μL.
4. Solution C (2×): Two tubes, 1.5 mL each
5. CyQuant GR Dye (400×): One tube, 50 μL.
6. Retrovirus RNA Standard: One tube, 500 μL containing 200 μg/mL retrovirus RNA Standard

Storage

Store all kit components at 4°C

General Description

Retroviral gene transfer is a technique for efficiently introducing stable, heritable genetic material into the genome of any dividing cell type. Replication-incompetent retrovirus is usually produced through transfection of the retroviral vector into a packaging cell line. Retroviruses are classified according to the receptors used to enter host cells. Ecotropic virus can recognize a receptor found on only mouse and rat cells. Amphotropic virus recognizes a receptor found on a broad range of mammalian cell types.
The murine leukemia virus (MMLV)-based vector is the most widely used retroviral vector in gene therapy due to its ability to stably integrate its transgene into host chromosomal DNA with low immunogenicity. The titration method to determine viral titer, which is performed by overlaying viral supernatant onto target cells (e.g., NIH 3T3 cells) after serial dilution, is widely used to represent infectious viral concentration as number of colonies per volume (i.e., CFU per milliliter). However, this colony-forming assay is time consuming (7 days or more). In addition, the titers determined by different groups can vary due to inconsistent conditions used for the same titration method such as target cell type, target cell number, polycation (e.g., Polybrene) concentration, incubation temperature, and exposure time for transduction.
The Retroviral Quantitation Kit does not involve cell infection; instead it specifically measures the viral nucleic acid content of purified viruses or unpurified viral supernatant sample. In the case of unpurified viral supernatant, the kit is especially useful for determining the supernatant titer before the transduction step. The kit has detection sensitivity limit of 1.5 X 10⁹ Viral Particles (VP)/mL, which is sufficient for mid or high-titer retrovirus sample. The entire procedure takes about 60 minutes. Each kit provides sufficient reagents to perform up 20 tests.
Retroviral Quantitation Kit provides an efficient system for rapid quantitation of retrovirus titer for both viral supernatant and purified virus.

Standard Curve

The following figures demonstrate typical quantitation results. One should use the data below for reference only. This data should not be used to interpret actual results.

Figure 1: Retrovirus RNA Standard Curve. The Retrovirus RNA Standard was diluted as described in the above instructions. Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer (Molecular Devices) with a 485/538 nm filter set and 530 nm cutoff.