Dual 96 well geneamp pcr system 9700

Tumor and matched peripheral blood samples from 97 patients were obtained from the endocrine-related tumor bank of Shanghai Key Laboratory for Endocrine Tumors. The genomic DNA was prepared using the QIAGEN DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Written informed consents were obtained from all of the study participants, and the protocols were approved by Ruijin Hospital Ethics Committee, Shanghai Jiao Tong University School of Medicine. The polymerase chain reaction (PCR) was performed in a Dual 96-well GeneAmp PCR system 9700 (Applied Biosystems, Courtaboeuf, France) using 20 ng of template DNA from each frozen sample per reaction. The products were sequenced by a 3730xl DNA analyzer (Applied Biosystems, Courtaboeuf, France). All of the sequences were analyzed using sequencing analysis software version 5.2 (Applied Biosystems, Courtaboeuf, France). The tumor samples had been screened for mutations in hotspot areas of PRKACA and GNAS and in the whole exomes of PRKAR1A.

The identified potentially pathogenic germline variants were validated by Sanger sequencing after PCR amplification. The PCR primers were designed using the Primer3 software package [41 (link)] (primer sequences available upon request). The PCR reactions were performed using a Dual 96-Well GeneAmp PCR System 9700 (Applied Biosystems) using standard protocols. Mutation analyses were performed using the Vector NTI software package (Invitrogen, Paisley, UK).