Dna specimen diluent from the cobas 4800 mutation test kit

Hematoxylin-Eosin (HE)-stained slides of CRC tissues were reviewed by a pathologist (H.S.L). Tumor areas were identified and microscopically dissected to sections with an area of more than 1 × 1 cm and comprising more than 60% tumor cells. One or two 8-μm-thick formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were de-paraffinized in xylene for 5 min at room temperature (RT), dehydrated in absolute alcohol for 5 min at RT, and air dried completely for 10 min. DNA was isolated using the Cobas DNA Sample Preparation Kit (Roche, Branchburg, NJ, USA) according to manufacturer's instructions, and the same preparation protocol was followed for all Cobas mutation kits used in this study. The concentration of the isolated DNA was measured using a NanoDrop UV spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and the DNA was diluted with DNA Specimen Diluent from the Cobas 4800 Mutation Test kit (Roche) to the optimal concentration for each gene (KRAS, 4 ng/μL; BRAF, 5 ng/μL; and PIK3CA, 2 ng/μL). Amplification and detection were performed using an Automated Cobas X480 analyzer. The real-time PCR assay was performed to detect the mutation in codons 12, 13, and 61 of KRAS; the V600E BRAF mutation; and the mutation in exons 1, 4, 7, 9, and 20 of PIK3CA.

Tumor samples were collected from surgical resection specimens of the primary CRC. Hematoxylin-Eosin (HE) stained slides were reviewed by a pathologist (H.S.L). Tumor areas were identified and microscopically dissected more than a 1 x 1 cm area, which consisted of more than 60% tumor cells. One or two 8-μm-thick formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were deparaffinized with xylene for 5 min at room temperature (RT), dehydrated in absolute alcohol for 5 min at RT, and allowed to air dry completely for 10 min. DNA was isolated using the Cobas DNA Sample Preparation Kit (Roche, Branchburg, NJ, USA) and the same preparation protocol for all Cobas mutation kits was used in this study. The concentration of the isolated DNA was measured using a NanoDrop UV spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the DNA was diluted with DNA Specimen Diluent from the Cobas 4800 Mutation Test kit (Roche) to the optimal concentration for each gene (KRAS 4 ng/μL, BRAF 5 ng/μL, and PIK3CA 2 ng/μL). Amplification and detection were performed with an Automated Cobas X480 analyzer instrument. The real-time PCR test could detect codon 12, 13, and 61 of KRAS mutation, V600E BRAF mutation, and exon 1, 4, 7, 9, and 20 of PIK3CA mutation.