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The BRAF V595E (qPCR) product is used for the quantitative determination of copies of the BRAF gene carrying the V595E mutation in total DNA preparations obtained from dog urine or extracellular DNA preparations obtained from dog blood.
Kit size: 100 reactions
Reaction: duplex (FAM: BRAF gene carrying the V595E mutation, HEX: internal control)
Internal control: exogenous/endogenous
Determination: qualitative/quantitative
Kit components:
The BRAF V595E (qPCR) product is intended for the analysis of the V595E mutation within the BRAF gene. This gene encodes the B-Raf serine/threonine protein kinase. The presence of this mutation has been reported in over 80% of cases of transitional cell carcinoma (TCC) in dogs. At the DNA level, the mutation involves the replacement of the thymidine (T) nucleotide with the adenine (A) nucleotide.
The product can be used to detect cancer cells in test material in the form of urine, biopsy, and blood samples. The kit is quantitative. It is used to determine the percentage of cells carrying a mutated copy of the BRAF gene in relation to the total pool of cells present in the test material. In the case of blood samples, the analysis is based on determining the proportion of DNA molecules released from circulating tumor cells (ctDNA) present in the entire pool of so-called circulating cell-free DNAs (cfDNA).
The BRAF V595E (qPCR) product is based on quantitative measurement of the number of copies of the BRAF gene carrying the V595E mutation and the number of copies of the B2M gene. The number of copies of the mutated version of the BRAF gene reflects the number of cancer cells present in the tested material. The number of copies of the B2M gene corresponds to the total number of dog cells present in the tested material.
The kit contains two pairs of primers. One pair of primers amplifies the mutated version of the BRAF gene. The resulting PCR product is detected using a Taqman probe labeled with FAM dye. The second pair of primers amplifies a fragment of the B2M gene. The resulting PCR product is detected using a probe labeled with HEX dye.
Quantitative determination of the number of detected DNA fragments is possible thanks to a standard curve prepared on the basis of calibrators included in the kit. To quantitatively determine the percentage of cancer cells, use the appropriately designed spreadsheet provided with the kit. The presence of cancer cells above 0.5% indicates the presence of a cancerous process. The threshold is based on publication Tagawa M., “Quantitative analysis of the BRAF V595E mutation in plasma cell-free DNA from dogs with urothelial carcinoma,” 2020, PLoS One;15(4):e0232365. A value above 0.3% is significantly higher than the background level (p-value < 0.001).
