Qiamp ffpe dna kit

We extracted DNA and RNA from formalin-fixed, paraffin-embedded tissues. After hematoxylin and eosin (H&E)-stained slide review and tumor tissue selection, we manually microdissected the corresponding tissue into 20 unstained, 5-μm-thick tissue sections (10 slides for DNA sequence and 10 for RNA). We purified DNA from the sample using a QIAmp FFPE DNA kit (Qiagen, Germany). For whole-exome sequencing, a minimum of 100 ng of DNA was used for the sequence library. DNA quality was determined by Caliper BioAnalyzer 2100 Instrument (Agilent Technologies, Santa Clara, CA, USA) and was confirmed by real-time PCR before sequencing. DNA samples were submitted to WuXi NextCode for next-generation sequencing using the low-input Agilent SureSelect V6 Kit. Paired-end sequencing (2 × 150 bp reads) was performed on successful DNA libraries using an Illumina HiSeq X-Ten after whole-exome capture. For RNA sequencing, we purified RNA with RNeasy FFPE kit from FFPE slides. RNA quality was determined with DV₂₀₀ value (DV₂₀₀ > 30%) by Caliper BioAnalyzer 2100 Instrument. RNA samples were also submitted to WuXi NextCode for next-generation sequencing with TruSeq RNA Exome. Paired-end sequencing (2 × 150 bp reads) was performed on successful RNA libraries using the Illumina HiSeq X-Ten as mentioned above.

Formalin-fixed paraffin-embedded tumour tissues were reviewed for quality and tumour content. A tissue containing at least 80% of neoplastic cells was selected for each case. Macrodissection of 7 μm methylene blue-stained sections allowed the separation of neoplastic and normal cells. Genomic DNA was extracted using the Qiamp FFPE DNA kit (Qiagen, Chatsworth, CA, USA) following the manufacturer's instructions. Mutational analysis of KRAS exons 2, 3 and 4 was performed as previously described [13] (link), [14] . KRAS exon 2 status was further confirmed through a specific mutant enriched polymerase chain reaction (PCR), known to be a more sensitive approach [15] (link). BRAF (exon 15), NRAS (exons 2 and 3) and PI3KCA (exons 9 and 20) mutational analysis was performed by means of PCR using specific primers previously described [13] (link), [15] (link). The PCR products were subjected to direct sequencing using an ABI Prism 3500 DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and then evaluated by means of the ChromasPro software.