Human FGFR3 Gene Test Kit
This product is used to qualitatively detect 6 hotspot mutations on human FGFR3 gene in RNA of formalin-fixed paraffin-embedded samples of bladder cancer patients; using fluorescent PCR technology, the test report can be obtained on the same day to meet the clinical demand for timely diagnosis and treatment, and assist clinical realization of individualized tumor treatment.
This kit is based on the principle of amplification refractory mutation system (ARMS) combined with fluorescence quantitative PCR technology for qualitative detection of RNA extracted from paraffin-embedded samples. The basic principle of ARMS-PCR is that Taq DNA polymerase lacks 3′-5′ exonuclease activity and Taq DNA polymerase cannot be extended if the bases at the 3′ end of the primer are not complementary to the template bases. The kit is designed with specific mutation template complementary primers based on known mutations, so that the 3′ end base is fully complementary to the mutation template and not complementary to the wild-type template, and at the same time, one or two mismatch bases are introduced at the 2nd-5th base of the 3′ end of the primer to form multiple mismatches with the wild-type template to prevent the extension of the wild-type template, which enables the detection of FGFR3 mutated gene.
The detection principle of the two fusion sites in this kit is to design specific primers and taqman probes on the exons of the two fused genes respectively, and combine with fluorescence quantitative PCR technology to qualitatively detect the RNA extracted from paraffin-embedded samples.
A total of three fluorescently labeled probes, FAM, CY5 and VIC, were used in this kit. The probe for detecting point mutations was fluorescently labeled with FAM, the probe for fusion sites was fluorescently labeled with CY5, and the internal control was fluorescently labeled with VIC. The internal control reagents monitored the quality of sample RNA extraction and reverse transcription and other processes. The kit also contains UNG enzyme, which can selectively degrade PCR products containing dU and effectively reduce false positives caused by PCR product contamination. A total of 6 reaction wells are required for each sample, and only a small amount of extracted sample RNA needs to be added to each reaction well. The quality of the sample is judged by the CT value of the internal control, and the CT value of the sample reaction wells is used to judge the negative positivity of the sample.
1. Extract RNA from formalin-fixed paraffin-embedded samples from bladder cancer patients.
2. Prepare the reverse transcription reaction according to the instructions.
3. Add the extracted RNA, positive quality control and negative quality control to the corresponding eight-linked tube positions.
4. Perform qPCR instrument loading experiments.
5. Interpret the results according to the internal control CT value and sample CT value