Picopure

For LCM, 5-µm sections of FFPE tumor tissue were cut and stained with H&E for histopathologic assessment and confirmation of diagnosis. Once confirmed, 10-µm sections were cut and placed on Leica Microsystems (Wetzlar, Germany) RNase-free polyethylene naphthalate (PEN)-membrane slides. The resulting FFPE sections were stained with H&E and dehydrated in 100% ethanol (Histogene LCM Staining Kit, Life Technologies, Grand Island, NY). LCM was performed using a Leica Gravity, contact-free collection system (LMD 6500). Isolated tumor cells were dropped immediately into PicoPure® (Life Technologies) DNA extraction buffer and incubated at 42°C for 30 min. Following incubation, samples were stored at −80°C until the time of DNA isolation. To assess Bap1 allelic loss in LCM-isolated tumor cells from a spontaneous MM, DNA was extracted using an AllPrep DNA/RNA FFPE Kit from Qiagen (Valencia, CA). Matching tumor and tail DNA were used as templates to amplify a portion of the mouse Bap1 gene in the region encompassing exons 6 and 7, using PCR with primers previously described for genotyping purposes (16 (link)). The Biorad Quantity One program was used to quantitate the intensity of the larger WT (634 bp) Bap1 allele PCR product and the smaller knockout allele PCR product (158 bp). The ratio of WT to mutant band intensities was then determined for each sample.