The Alu HIV PCR is the method of choice for quantification of integrated HIV DNA in cellular genomes see also
(Liszewski et al., 2009) for an in depth protocol of the method.
by performing a repetitive sampling strategy with more than 40 replicate PCR reactions, the data can be assessed in a similar manner as digital PCR data.
To enable data analysis of this repetitive sampling Alu-HIV PCR method we provide
an Excel template
in which the raw quantitative output of the Alu-HIV PCR should be inserted as described previously
(De Spiegelaere et al., 2014).
The excel template
composed for Alu-HIV PCR quantification: the colored cells in the lower left quadrant require data input,
i.e. the correction factor as measured on the standard for calibration to assess the fold difference between
the observed and expected number of integrations/well, the total number of Alu-HIV replicates (required for accurate error estimation)
the number of cells per replicate reaction (assessed by a qPCR on a reference gene, e.g. RPP30),
and the raw quantification cycle (Cq)* values of the Alu-HIV PCR and the HIV only PCR. Note that negative reactions should be left empty
not to interfere with the analysis. After data input the results are shown in the shaded areas of the lower right quadrant,
showing the amount of proviral HIV per well, per cell or per million cells along with the confidence intervals of each estimate.
*note that the Cq values are in some real-time PCR programs referred to as treshold cycle (Ct), crossing point (Cp) or take-off point (TOP)
Please cite the following reference when using this method:
De Spiegelaere W., Malatinkova E., Lynch L.,Van Nieuwerburgh F.,Messiaen P.,O’Doherty U. and Vandekerckhove L. (2014)
Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics.
Clinical Chemistry published March 24, 2014 as doi:10.1373/clinchem.2013.219378