The samples were then incubated for another 30 min, whereupon TrxR activity was measured in both samples using the direct DTNB assay [50].
Correlations between the D1 and D2 scales were also calculated for both samples using the Pearson product moment coefficient.
Hardy-Weinberg equilibrium (HWE) was tested in both patient samples using the chi square test.
Cyanine 3-CTP (Agilent protocol -labeled cRNA targets were protocol -labeled ng of total RNA from both experimental samples using the Agilent One-cRNAr Microarray-Based Gene Expression Analysis kitargetsent Technologies, Santa Clara, CA, USA).
We also calculate the carrier tunneling time for both samples using a modified form of the semiclassical Wentzel Kramers Brillouin (WKB) approximation [26 28].
We cloned both loci from all samples using the pGEM-T Easy Vector System I (Promega, Madison, Wisconsin) and following the protocol of [ 73] for cloning, colony selection, and post-cloning re-amplification with universal M13 primers.
PCR was performed for both tumor and normal tissue samples using the following program: 95°C for 15 minutes, followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec.
Genomic DNA was extracted from both field and museum tissue samples using the QIAamp™ DNA Mini Kit for DNA purification (QIAGEN) following standard protocol.
Additionally, both laboratories had assayed the sample using the same protocol, suggesting that differences in technique and timing, though impossible to eliminate, should have been minimal.
To mitigate the effect of these caveats, biological validation, such as qPCR, would normally be performed using the same DNA samples but was not feasible in this study, both in acquiring the same samples used for the study (as it has been shown that the strains may not be isogenic, and are subject to somatic mosaicism), as well as the scale of the project.
