Magcore nucleicacid extractor

Genomic DNA was extracted from 5 to 10 μm of FFPE tissue using a DNA extractor (MagCore NucleicAcid Extractor, RBC Bioscience, Taiwan) and MagCore Genomic DNA FFPE One-Step Kit, following the manufacturer’s recommendations. Genomic DNA quality was assessed using BIOMED-2 control gene PCR protocol and samples with a DNA product size of ≥ 300 base pairs (bp) were analyzed [11 (link)]. Before initiating VDJ gene rearrangement analysis by HTS, all cases were analyzed to evaluate clonality according to the BIOMED-2 protocol [11 (link)]. NGS analysis was performed on 454 GS Junior system (Roche) previously described [12 (link)]. Data analysis was performed using the Roche (Basel, Switzerland) proprietary software package for the 454 GS Junior system (Roche). Image acquisition, image processing, and signal processing were performed during the run.

All sample were analyzed at the Fondazione Policlinico Universitario Agostino Gemelli IRCCS in Rome, Italy. Blood or buccal swab samples were collected for each participant. Genomic DNA was extracted from blood or buccal swab samples using the automated MagCore Nucleic Acid Extractor (RBC Bioscience Corp., New Taipei City, Taiwan) instrument, in association with MagCore^® Genomic DNA Whole Blood Kit. DNA concentration was determined using the NanoPhotometer P-Class (Implen, Westlake Village, CA, USA). Analysis by PCR was conducted using primers showed in Table 1. PCR and Sanger sequencing were used for MCT1 and COL1A1 genotyping. In detail, PCR products were sequenced on the 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in association with SeqScape™ software. For ACE (I/D) genotyping, PCR and 4% electrophoresis gel were used. PCR/RFLP fragments analysis was used for ACTN3 R577X genotyping. ACE and ACTN3 genotype assignment was determined using the Invitrogen™ iBright™ CL1500 Imaging System according to the expected amplicon patterns.