KSHV DNA sequence (ORF 26) is detectable in MM BMSCs. Ficoll Hypaque BMMCs were freshly obtained from 26 patients with MM and 4 normal donors and cultured for 2 to 6 weeks in Iscove’s media with 10% FBS, 10% horse serum, and penicillin/streptomycin to generate long-term BMSC cultures. Adherent layers were trypsinized and assayed for presence of KSHV DNA sequence. (A) DNA from long-term BMSCs from 12 patients with MM and 2 normal donors were assayed by PCR using primers that amplify KSHV gene sequences (KS330₂₃₃) followed by nested PCR using primers designed to recognize sequences internal to KS330₂₃₃ to yield a final PCR product of 186 bp. DNA from BCBL-1 cell line (lane 1) served as a positive control for the detection of KSHV gene sequences and DNA obtained from normal BMSCs (lanes 2 and 3) as a negative control; MM patient BMSCs are shown in lanes 4 through 15. (B) Amplification with β-actin specific primers confirmed DNA in each lane. (C) To confirm the specificty of PCR, nested PCR products were transferred onto nitrocellulose filters. Southern blot analysis was performed by hybridizing the filters with a³²P end-labeled 25-bp oligomer probe internal to the KS330₂₃₃ sequence.