title: QIAGEN Therascreen KRAS RGQ Assay, QIAGEN KRAS Pyro Assay, and Dideoxy Sequencing for Clinical Laboratory Analysis of KRAS Mutations in Tumor Specimens.
abstract: OBJECTIVE
  To compare the performance of assays used to assess KRAS mutations in tumor specimens.
  
  
  METHODS
  We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method.
  
  
  RESULTS
  We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86% for the RGQ assay compared with 100% for the Pyro but 100% for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ.
  
  
  CONCLUSIONS
  The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE.
authors.forename: Qamra M
authors.initials: QM
authors.lastname: Alqahtani
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE.
authors.forename: Ann
authors.initials: A
authors.lastname: Crowley
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE.
authors.forename: Sharleen
authors.initials: S
authors.lastname: Rapp
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE acushman@unmc.edu.
authors.forename: Allison M
authors.initials: AM
authors.lastname: Cushman-Vokoun
mesh_terms: D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000473:pathology; D057089:Pathology, Molecular / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: DNA; EGFR inhibitor; biomarker; colorectal cancer; formalin-fixed paraffin embedded; pyrosequencing; real-time PCR; targeted therapy