Standard curve and proviral load interpolation

For routine quantification of DNA or RNA, two general methods of data analysis exist: absolute quantification and relative quantification. Absolute quantification determines the exact template copy number, usually by relating the PCR signal to a standard curve. So it is important to know what kind of a template for a standard curve is used. In general, two kinds of control template—a plasmid vector inserting the region of HTLV-1 genome, and an HTLV-1-infected cell line monoclonally integrating one copy of the provirus for generation of a standard curve—are used. Then, unknown copy numbers are calculated by interpolation from such plasmid control regression curve and reported as copy equivalents per 104-6 PBMCs or percent. In our case, an HTLV-1 standard curve is generated using serial dilutions as the template for the real-time PCR from 107 to 101 copies of the proviral DNA derived from a clone in pBR322 vector as follows. A 9-kb proviral DNA fragment is cloned into the pBR322 and propagated. After digestion with EcoRI, the insertion is purified and quantified with the PicoGreen dsDNA Quantification kit. Molecular concentrations are calculated and the purified insert is used for the generation of the standard curve (Fig. 1).

The copy numbers in the samples are estimated by interpolation from the plasmid control regression curve and an internal control of p-globin to correct for sample DNA quality differences. Copy numbers were reported as copy equivalents per 104 PBMCs, mainly lymphocytes.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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