Hi, if you drop me an email (on my web page at www.genetherapy.science) I'll put you in touch with one of my PhD students to send over a protocol.

It cannot be put into any better words or context than Jochen has just stated. Attached here is a 2010 publication to back his statements up as well.

An additional consideration here is to realize that the linearized plasmid containing the target sequence, and the endogenous experimental sample target sequence, being two differently-derived sample preps, will still be expected to amplify at different amplification efficiencies.  One must reconcile this mathematically.  I suggested a way to do this in a 2011 book chapter.  The equation I proposed is attached here. I have it as a tattoo on my shoulder. (Well...maybe next year anyway).

The lingering detail in this issue, is to be sure to account for the threshold used on the machine.  If a different threshold is used for the linearized plasmid target standard curve and the experimental sample target amplifications, the result of the right side of the Xo equation must then be multiplied by the quantity (threshold adjustment factor): 

10(Experimental sample target threshold - Linearized plasmid target threshold)

This is the pineapple-topper for this relationship.

Underlying this is the interesting realization that every single amplification sigmoidal curve (when y-axis is in log scale and x-axis is in simple progressive integer Cq scale) is a standard curve in and of itself.  When one rotates the amp plot image 90 degrees clockwise, the slope of the log-linear region of the amplification curve itself, if a 'perfect' amplification, should be -3.3219 (indicating 100% amplification efficiency of that target).  The perfect threshold is theoretically drawn through the best inflection point of the sigmoidal curve - and can indeed be a different point for each curve - such as the Cy0 Method (by Guescini and Sisti) illustrates/suggests.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and engrossed in the intricate world of genetic research. Your inquiry regarding the quantification of lentivirus integration or copy number in transduced cells via quantitative reverse transcription PCR (qRT-PCR) is both pertinent and challenging. Accurately determining the integration or copy number of a lentiviral vector within a host genome is crucial for assessing the efficiency of transduction and understanding the potential for gene expression. Below, I outline a methodical approach for employing qRT-PCR to quantify lentiviral integration in transduced cells.

Overview of the Approach

Quantifying lentivirus integration using qRT-PCR involves designing primers specific to a sequence within the lentiviral vector and a reference gene within the host genome. The comparison of the amplification of these two targets allows for the determination of the lentiviral copy number per cell.

Step-by-Step Protocol

Considerations and Troubleshooting

• Primer Specificity: Ensure the specificity of the primers to prevent amplification of similar sequences, which could lead to inaccurate quantification.
• Efficiency of Transduction: The method assumes that the reference gene is present in one copy per genome. Adjustments may be needed for polyploid cells or cells with genomic aberrations.
• Sensitivity and Dynamic Range: Validate the sensitivity and dynamic range of the qRT-PCR assay to accurately quantify low levels of lentiviral integration.

By meticulously following this structured approach, you can accurately quantify the integration or copy number of lentiviral vectors in transduced cells using qRT-PCR. This quantification is invaluable for assessing the efficiency of lentiviral transduction and for further genetic analyses.

Should you require further guidance or have additional questions, please do not hesitate to reach out. Your dedication to advancing our understanding of lentiviral vectors is highly commendable.

Warm regards.

This protocol list might provide further insights to address this issue.