Cobas dna sample preparation kit

For all suitable tumour specimens, 5 and 2 μm tissue section slides were prepared. The 5 μm slides were used for DNA extraction and subsequent analysis for EGFR, KRAS, BRAF and NRAS while the 2 μm slides were used directly for fluorescent in situ hybridisation (FISH) analysis of ALK and MET. DNA extraction was done using Roche cobas^® DNA sample preparation kit (Roche Molecular Systems Incorporation, Branchburg, New Jersey, USA).

The EGFR PCR test (cobas EGFR Mutation Test, Roche Molecular Systems, Inc, Branchburg, NJ, USA) is a CE-IVD marked multiplex allele-specific PCR-based assay designed to detect 41 mutations in exons 18, 19, 20, and 21 in FFPET specimens of human NSCLC.[28] (link) DNA is isolated using the cobas DNA Sample Preparation Kit (Roche Molecular Systems, Branchburg, NJ). [29] A minimum of 150 ng of genomic DNA is required for PCR amplification, which can typically be isolated from a single 5 µm FFPET section. The EGFR PCR test software version used in this study was designed to detect 29 deletions in exon 19 and 2 L858R variants in exon 21. Macrodissection is only recommended if tumor content is less than 10%; laser capture microdissection is not required. The EGFR PCR test was performed per manufacturer's package insert and results were automatically analyzed and reported. The limit of detection has been validated to 5% mutant alleles. The workflow from DNA isolation to results reporting can be performed in one 8 hour period.[27]

One slide at the beginning of each serial section was stained with hematoxylin-eosin and histopathologically examined to determine the tumor cell content. Only samples with a tumor cell content of at least 30% were included in this study. The area of interest was circled on the stained slide and macrodissection was performed on the corresponding unstained slides using a scalpel. DNA was extracted using the cobas^® DNA Sample Preparation Kit (Roche, Grenzach-Wyhlen, Germany).

Quality and tumour cell content of the FFPE tissue block sections were evaluated by an experienced pathologist. Genomic DNA was extracted from representative FFPE tissue blocks, containing at least 50–70% neoplastic cells. In cases of low tumour tissue content, a macrodissection was performed to enrich the amount of tumour cells. Genomic DNA was extracted from a 5 μm thick section of MM tissue, using the Cobas^® DNA Sample Preparation Kit (Roche) according to the manufacturer’s instructions. In CRC samples, genomic DNA was extracted from six 7 μm thick serial FFPE sections. DNA extraction was performed using the QIAmp Mini kit (Qiagen) according to the manufacturer’s instructions.

Preoperative biopsy or postoperative tumour specimens were used for KRAS gene detection. Tumour tissues were fixed in 10% neutral buffered formalin, processed, and then embedded in paraffin for light microscopy. The sections were stained with haematoxylin and eosin (H&E) for histological examination. The Cobas DNA sample preparation kit was used to extract DNA from formalin-fixed paraffin-embedded tissue sections (Roche Molecular Systems, Inc., Branchburg, NJ, USA) according to the instructions, and the reaction was carried out with the Mx3000PTM real-time PCR system (Stratagene, La Jolla, USA). Using a real-time polymerase chain reaction assay, the Cobas KRAS Mutation Test (Roche Molecular Systems, Inc.) and LightMix KRAS and NRAS kits (Roche Molecular Systems, Inc.) were applied to detect KRAS mutations. Tumours harbouring KRAS mutations in either preoperative biopsy or post-treatment resection specimens were considered KRAS mutants.