Pyromark kras kit

The analysis of KRAS mutation status was performed by using PyroMark Q24 KRAS v2.0 assays to detect KRAS codons 12, 13, 61 and 146 mutations. In brief, 2–10 ng of genomic DNA was subjected to PCR reactions. Twenty μl of PCR product was subjected to Pyrosequencing analysis on a Q24 instrument (QIAGEN) by using the CE-IVD marked PyroMark KRAS kit (QIAGEN) according to the manufacturer’s instructions.

For KRAS mutation assay, genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor sections using QIAmp Kit (QIAGEN, Valencia, CA) and amplified by polymerase chain reaction. DNA pyrosequencing was performed using PSQ HS 96 Gold SNP Reagents (Biotage, Uppsala, Sweden) with a PSQ HS 96A Pyrosequencer. Separate assays for detection of codons 12/13 and codon 61 were performed, using primers from the PyroMark KRAS kit (QIAGEN, Valencia, CA).
Expression of MMR proteins (MLH1, PMS2, MSH2, and MSH6) was analyzed in formalin-fixed, paraffin-embedded tumor sections using immunohistochemistry. Monoclonal antibodies included anti-MLH1 (clone G168-728), anti-PMS2 (clone MRQ-28), anti-MSH2 (clone G219-1129), and anti-MSH6 (clone 44). MMR protein loss was defined as the absence of nuclear staining in tumor cells in the presence of positive nuclear staining in normal colonic epithelium and stromal cells. Tumors were defined as MMR-deficient (dMMR) if one or more MMR proteins was lost, and MMR-proficient (pMMR) if all MMR proteins were detected.