Dolph Houben

121 Transplant chimerism in bone VCA 6 Whole tissue DNA/RNA extraction Genomic DNA (gDNA) was extracted from bone, liver, and spleen of each animal. Frozen bone sections were pulverized in liquid nitrogen using a bone mill (A11 basic analytical mill, IKA-Werke GmbH & Co. KG, Germany). Liver and spleen samples were pulverized in liquid nitrogen using a mortar and pestle. Approximately 10mg of tissue was used for extraction, using a DNeasy Blood&Tissue kit (Qiagen, Cat. No. 69504, Hilden, Germany). RNA was extracted from the pulverized bone with a PureLink RNA mini kit, TRIzol reagent and on-column Pure link DNase treatment (Thermo Fisher Scientific, Cat no. 12813018A, 12034977, 12185-010, Carlsbad, CA.). Quantification and determination of both gDNA and RNA purity were performed with a Nano-drop Spectrometer (ThermoScientific Nano-drop Technologies, Wilmington, DE). The absence of RNA degradation was confirmed by gel electrophoreses of the total RNA before conversion to copy DNA (cDNA). iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA) was used for the reverse transcription reaction. Total RNA (200ng) was mixed with nuclease-free water, iScript reverse transcriptase, and 5X reverse transcription reaction mix and converted to cDNA following iScript manufacturer’s protocol. We also extracted gDNA from female porcine bone and male porcine bone. These were used along with commercially available purified male and female porcine gDNA (BioChain Institute Inc., Newark, CA) for use as control samples. All gDNA and cDNA samples were stored at -20⁰C until PCR was performed. Quantitative Real-Time Polymerase Chain Reaction Real-time qPCR was carried out to evaluate the extent of transplant chimerism as well as systemic microchimerism. Two separate analyses were performed in the VCA segment: newly formed bone obtained by LCM, as well as an entire cross-section of the transplant. We analyzed the ratio of male gDNA to an autosomal housekeeping gene (RPL4) , expressed in both male and female cells. A similar analysis of extracted RNA demonstrated relative gene expression from viable male (allogeneic) and female (autogenous) cells. Different primer sets were designed for gDNA analysis and RNA analysis (Table 1). The SRY -gene is located on the Y-chromosome and is therefore used to detect recipient or donor-specific cells in sex-mismatched models [13, 14] . Ribosomal protein L4 ( RPL4 ) is a commonly used housekeeping gene in porcine models [15] . SRY primer sets (Integrated DNA Technologies, Coralville, IA) were designed for both the intronic and the exonic sequences of SRY. The intronic primer set was used to detect male cells (gDNA) and the exonic primer set ( SRY -e) was used to detect RNAs produced by viable male cells [16] .