In the study of Shen and Qin a p. V600K mutation was overlooked selleck chemical Alisertib by visual inspection but was detected using pyrosequencing data analysis soft ware. Using software tools and a customer designed assay set up can avoid such problems. Besides, it allows the detection of a broader spectrum of mutations and reduces the costs down to one quarter. Allele specific PCR The cobas 4800 BRAF V600 test is the only CE IVD marked test used in this study. The CE IVD mark indi cates that this test meets essential requirements regarding safety, health and environmental protection. 60 out of 82 tumor samples were analyzed with the cobas BRAF V600 test. All samples showed a valid result. The allele specific PCR used in this test generates an amplicon of 116 base pairs containing codon 600 in exon 15 of the BRAF gene.

Amplification curves are Inhibitors,Modulators,Libraries shown only for the mutant and the wildtype control but not for the samples analyzed and a non template control is not provided. Data are analyzed when mutant and wildtype Inhibitors,Modulators,Libraries controls have a valid status. A re port is generated automatically Inhibitors,Modulators,Libraries and results can be distin guished between mutation detected and mutation not detected. This test is specific for the p. V600E mutation with a reported sensitivity of 5% mutated alleles in a background of wildtype alleles. Limit of detection in our preselected cohort was 7% mutant alleles in a back ground of wildtype alleles. 36 of 37 p. V600E mutations were detected with the cobas BRAF V600 test. One case with a border line frequency of 5% of mutated alleles using pyrosequencing could not be detected.

But it should be taken into account that we extracted the DNA with our standard in house method and not with the recommended kit. This may influence the test results. Furthermore, the marked area on the HE stained slide contained many lymphocytes diluting the p. V600E alleles. Curry et al. showed Inhibitors,Modulators,Libraries an even lower limit of detection of 4. 4% mutated alleles per 1. 25 ng/ul on FFPE tissues for the p. V600E mutation. In contrast, Lade Keller et al. performed a dilution series of p. V600E mutated DNA followed by analysis on the cobas 4800 BRAF V600 test. This test was not able to detect a p. V600E mutation on the dilution point that theoretically Inhibitors,Modulators,Libraries contained 10% mutant alleles. Analysis have shown cross reactivity with p. V600E2, p. V600K and p. V600D but not with p. V600R mutation.

In our cohort, the www.selleckchem.com/products/chir-99021-ct99021-hcl.html cobas BRAF V600 test showed cross reactivity five times in p. V600K mutated samples containing 59, 61, twice 62 and 64% of mutated alleles using pyrosequencing. One p. V600K mutation with a frequency of 57% that is above the described cross reactivity, was not detected by the cobas 4800 BRAF V600 test. Furthermore, several additional cases with a mutation frequency below the described limit of detection were missed in our study case 9 showed a frequency of 6. 6% for p. V600K, case 36 25% for the same mutation and case 24 an allele frequency of 46% for the p. V600E2 mutation.