HAS gene expression in healthy donor bmMPCs and myeloma cells.

RT-PCR products were amplified by competitive RT-PCR and resolved on a 1.5% agarose gel. Band intensity was determined by densitometry as indicated in “Materials and methods.” (A) Expression of Has mRNA in bmMPCs: RT-PCR for Has1, Has2, and β-actin in 3 healthy donors and 3 multiple myeloma patients and done as described in “Materials and methods” using primers indicated in Table 1. A representative result of 3 experiments is shown. Expression of HAS mRNA in myeloma plasma cell lines and ex vivo bone marrow plasma cells: (lane 1) ANBL-6, (lane 2) U266, (lane 3) ARH77, (lane 4) RPMI 8226. RT-PCR for Has1, Has2, Has3, and β-actin was done as described in “Materials and methods” and using primers indicated in Table 1. β-Actin was amplified to determine that all samples contained equivalent amounts of cDNA. (B) Competitive RT-PCR for Has1 and Has2 in 3 multiple myeloma patients and 3 healthy donors. One microgram of total RNA was reverse transcribed as indicated in “Materials and methods”; 2.0 μL cDNA was subjected to competitive PCR using primers for Has1 and Has2 as indicated in Table 1. RT-PCR was carried out in duplicate. The graph in panel B shows the results from duplicate quantifications of Has mRNA from 3 myeloma and 3 healthy donors.