Fig. 4.

The measurement of a target MSRV sequence in mixed templates. Serially diluted standard templates were amplified in the presence or absence of ERVWE1-specific PNA during probe-based real-time PCR to measure the MSRV expression level. An amount of 1 × 10 e⁴ copies/reaction of plasmid DNA containing MSRV fragment (pSC-B-MSRV) was used as template and 0.4 μM of ERVWE1-specific PNA was added into the reaction mixture in this assay. The pSC-B-MSRV was then serially diluted with plasmid DNA that contained ERVWE1 sequence (pSC-B-ERVWE1). The proportions of pSC-B-MRSV were adjusted to 100, 80, 60, 40, 20, 10, 5 and 1 % (pSC-B-MSRV:pSC-B-ERVWE1 ratio). Control samples that did not contain PNA were analysed parallelly. In PNA-containing samples, a linearity of the plot was maintained in the whole range of MSRV concentration values but if no PNA was added into the reaction, a correct quantitation of MSRV was possible only if an amount of MSRV DNA was equal to 100 % in respect to ERVWE1 but not at the lower MSRV-to-ERVWE1 values when the measurement was not linear. The accuracy of the method was evaluated according to the calculated plasmid copy number (depicted as triangles at the plot). All samples were run in duplicates