The Adeno-X qPCR Titration Kit provides an extremely fast, simple, and accurate method for titrating adenoviral stocks from all Ad5-based adenoviral vectors. The kit delivers results in just four hours, a vast improvement over standard titration methods (such as the plaque assay), which require up to 10 days to complete. Because qPCR titration is so fast, target cells can be infected with accurately titrated virus on the same day the virus is harvested.
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Adenoviral titration by quantitative PCR
Rapidly determine adenoviral titers with our Adeno-X qPCR Titration Kit
- Harvest, titrate, and infect in a single day
- Measure titers from crude lysate or purified viral preps
The qPCR titration method
The protocol combines qPCR with TB Green chemistry, allowing you to determine the viral genome copy number in adenoviral preparations (i.e., crude lysates or purified stocks) from a calibrated DNA standard curve (Figure 2). The procedure is simple: viral DNA and control DNA (provided) are serially diluted and subjected to qPCR. The DNA copy number of each viral sample is then determined by comparing its Ct value to a standard curve generated by plotting the Ct values of the diluted control samples against their respective copy numbers (Figure 2). Titration assays demonstrating the consistency of this approach when used with either crude lysate or purified virus are shown in Figure 3.
Once the genome copy number of your viral stock is determined, it can be correlated with the number of viral infectious units (IFU; determined independently) to establish a copy number/IFU relationship (Table I). Determination of the copy number/IFU relationship for a given prep allows you to normalize the amount of the prep used in each experiment, for consistent interassay results.
|Table I: Comparison of Adeno-X qPCR titration to other titration methods|
|Titration method||Adeno-X qPCR titration ratios|
|Sample type||Virus||qPCRa (copies/ml)||Fluorb (IFU/ml)||X-gal (IFU/ml)||qPCR/Fluor (copies/IFU)||qPCR/X-Gal (copies/IFU)|
|Purified||AdAcGFP1||5.62 × 109||3.03 × 109||N/A||2||N/A|
|Prep A||1.44 × 1010||1.90 × 109||N/A||8||N/A|
|Prep B||1.46 × 1010||2.26 × 109||N/A||6||N/A|
|Prep C||1.38 × 1010||2.73 × 109||N/A||5||N/A|
|Purified||AdLacZ||1.01 × 1010||N/A||1.54 × 109||N/A||7|
|Prep A||1.33 × 1010||N/A||2.67 × 109||N/A||5|
|Prep B||1.24 × 1010||N/A||2.67 × 109||N/A||5|
|Prep C||1.21 × 1010||N/A||3.15 × 109||N/A||4|
- Adenoviral copy numbers were determined using the Adeno-X qPCR Titration Kit (Cat. # 632252).
- To determine fluorescence-based infectivity titers, adenoviral stocks were serially diluted (tenfold) and applied to HEK 293 cells. After 48 hr, fluorescent cells were scored with a fluorescence microscope.
Adenoviral selection guide
Browse our adenoviral selection guide to choose the best system for your needs. We offer both constitutive and Tet-inducible expression systems based on the Adeno-X adenoviral system 3, an advanced gene delivery system that quickly and efficiently produces adenoviral particles.Selection guide Product finder tool
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