Dolph Houben

123 Transplant chimerism in bone VCA 6 Data analyses The raw data from experimental samples produced by the CFX384 real-time detection system were transferred to Bio-Rad CFX Manager 3.0 software to calculate the efficiency curves. Transcript quantity measurements were normalized to RPL4 , using the 2^ (- ΔΔ CT) method [17] using Windows Excel. For DNA samples we refer to this ratio as the relative copy number of SRY . This ratio was measured for both male and female controls as well as the samples from both experimental groups. For RNA samples we calculated the relative expression ratio using the same method. In order to estimate the proportion of the allotransplant repopulated by female cells, we divided the relative copy number of SRY found in our samples by the mean relative copy number of SRY found in male control samples obtained from bone by the same method as the samples of interest and multiplied this by 100 ((Allotransplant copy number SRY ) / (mean copy number SRY male bone)) x 100)). Standard Curve A standard curve was run for SRY, SRY-e, and RPL4 using the synthetic amplicon to evaluate PCR efficiencies. The calculation of the standard curve was done by using linear regression analyses. A 10-fold serial dilution was used for the dilution of the amplicon, resulting in dilution from 10 1 -10 8 molecules/µL. Efficiencies ranged from 92.3% to 100%, coefficients 0.973-1.0 and standard curve slopes of -3.5. Statistics Since the data is collected from a low sample size, the Wilcoxon rank-sum test was used as a non- parametric test to detect a difference between the groups (allotransplants with Patent AV bundle versus allotransplants with ligated AV bundle and male control samples). All statistical tests were two-sided and differences were considered significant for p-values of < 0.05. Statistical analyses were performed using the statistical program JMP Pro 13.0.0 (SAS Institute Inc.) and GraphPad Prism 5.03 for illustrations (Graph Pad Software, La Jolla, CA). Statistical analysis was supported by the Center for Translational Sciences Activities (CTSA) at Mayo Clinic. Results All animals were full weight-bearing after an average of 4 days. Twenty weeks after transplantation all animals achieved complete union of the proximal host-transplant interface. Only three incomplete unions were observed at the distal host-transplant interface. Two pigs with surgical complications were excluded from the study. One sample was excluded from analyses due to degradation of the DNA and RNA. Leaving 6 animals in groups 1, and 5 in group 2. Quantitative real-time polymerase chain reaction analyses were carried out on male- and female-only gDNA control samples, to verify the specificity of the SRY primer sets and appropriate technique. The SRY/RPL-4 copy number ratios calculated demonstrated the designed SRY primer sets to be specific to male cells. Additionally, H 2 O-only samples were appropriately negative for DNA (Fig. 4). All extracted DNA and RNA samples expressed RPL-4 in our PCR reaction.