Geneamp 9700

The punched biopsy skin and muscle from the donor graft (female) and recipient tissues in the IR/rASC/FK group (male) were cut into small pieces, and around 1 mg of tissues was transferred to a 1.5 mL Eppendorf tube containing 100 μL of lysis buffer (10 mM Tris–HCl, pH 7.6, 10 mM KCl, 2 mM EDTA, 4 mM MgCl₂; all from Sigma-Aldrich) for genomic DNA extraction. The male-specific SRY gene to the donor allograft was amplified using the following forward primer set: Sus scrofa SRY_F (5′-CTG GGA TGC AAG TGG AAA AT-3′) and Sus scrofa SRY_R (5′-GGC TTT CTG TTC CTG AGC AC-3′). The SRY primers were designed from a sequence available on GenBank assembly ID: GCA_000003025.4. PCR amplification was performed under the following conditions: an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 20 s, annealing at 65 °C for 20 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 7 min in a thermal cycler (GeneAmp 9700; PerkinElmer, Foster City, CA, USA). All the amplified DNA samples were electrophoresed in a 1.5% agarose gel (Merck KGaA, Darmstadt, Germany) and visualized by ethidium bromide staining (Sigma-Aldrich) and photographed. The SRY protein profiles in donor allografts were determined by using IHC staining (Santa Cruz Biotechnology, Dallas, TX, USA).

DNA was isolated from 200 µL of serum by manual extraction using the QIAamp DNA Mini and Blood Mini kit (Qiagen, Venlo, The Netherlands) (samples from 2004 to 2006) or by automated extraction using the LC Nucleic Acid isolation kit (Roche, Almere, The Netherlands) (samples from 2007 to 2014). DNA was eluted in 50 µL of elution buffer and amplified in a nested PCR using a thermocycler (GeneAmp 9700, Perkin Elmer, Waltham, MA), as previously described.31, 35A 656‐nucleotide fragment was amplified, which encompassed the first 203 of the 226 amino acids of the HBsAg and amino acid 6 to 209 of the reverse transcriptase (RT) of the polymerase. The sequence reaction was performed in house or by BaseClear (Leiden, The Netherlands) with Big Dye Terminator (Life Technologies, Carlsbad, CA).