Qiamp system

Primary MM cells were stained with Hoechst 33342 and fluorescent-labeled CD138 antibody and SP and NSP/CD138+ cells were isolated by cell sorting using FACS Aria. DNA isolation was performed using the QIAmp system (Qiagen, Valencia, CA, USA), the resultant DNA concentration was estimated by NanoDrop ND-1000 spectrophotometer, and DNA quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). PCR primer sequences for IGH variable gene framework region 3 (FR3) analysis was designed as previously reported,^{23 (link)} and PCR was used to assess clonality using the BIOMED-2 system.^{24 (link)} PCR master mixes were purchased from Invivoscribe Technologies (San Diego, CA, USA), and PCR was performed as per the manufacturer’s instructions using HotStart Taq DNA polymerase (Qiagen).^{25 (link)}

DNA was isolated from cells using the QIAmp system (Qiagen). The concentration of DNA was estimated using the NanoDrop ND-1000 spectrophotometer. DNA quality was assessed using Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). PCR primer sequences for IGHV gene framework region 3 (FR3) were designed as previously reported (Kummalue et al, 2010 (link)). PCR of the IGH genes to assess clonality used the BIOMED-2 system. Master mixes were purchased from Invivoscribe Technologies (San Diego, CA, USA), and the PCR was done as per manufacturer’s instructions and used HotStart Taq DNA polymerase (Qiagen) (Burack et al, 2010 (link)).