A further evaluation of the quantification of the TNT-PNA conjugates was carried out to determine whether accurate relative quantification can be derived from TNT-PNA pairs with different PNA sequences, i.e. can a reference sequence in a sample probed with one TNT-PNA be used to quantify a second sequence with a different PNA probe as long as the TNTs used in the probe pair are isotopes of each other. This would enable quantification by internal calibration using spiked sequences, housekeeping genes or similar controls in quantitative expression profiling or diagnostic assays. In these experiments, a pair of biotinylated 50-mer target oligonucleotides was used (MOMP-50 again and a sequence from B. anthracis, ANTHRAX-50; see Table 3 ). These were captured onto streptavidin-coated magnetic beads. The quantity of one target was fixed while the relative quantity of the second was varied. The captured targets were then hybridized at room temperature with a probe solution comprising equal quantities of MOMP-12-TNT1 and Anthrax-12-TNT2 probes (see Table 2 ). Probes of the same length were used together. After the hybridization, the magnetic beads were washed as before and the hybridized TNT-PNAs were eluted from their targets on the beads into 50:50 methanol:water with 1% formic acid and analysed as described earlier. Typical results are shown in Figure 8 . As observed in the simple mixture experiments, the measured TNT ratios show a linear relationship with the expected ratios but the measured quantities do not exactly match the expected ratios when the TNT-PNA probes being compared are not true isotopes but the linear relationship does mean that the measurements are quantitative. These data suggest that with appropriate choice of reference sequences, quantitative internal calibration should be achievable, which would be very useful in situations where suitable reference samples are not available for external calibration.