Biomarkers for diagnosis of nasopharyngeal carcinoma and application thereof
A biomarker and nasopharyngeal carcinoma technology, applied in the field of tumor diagnosis, can solve the problems of slow development and unreported detection of nasopharyngeal carcinoma
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Embodiment 1
[0044] Microarray screening experiment of differentially expressed miRNA in saliva
[0045] 1. After obtaining the informed consent of the patients, saliva samples from 10 patients with nasopharyngeal carcinoma diagnosed for the first time and 10 healthy people were collected from Jiangsu Cancer Hospital as normal controls. The sampling time of saliva was based on 1 hour after the meal, and 2ml was reserved and stored in a -20°C refrigerator.
[0046] 2. Use the miRNeasy Serum / Plasma Kit kit (#217184, Qiagen, Hilden, Germany) to extract RNA from the saliva of patients with nasopharyngeal carcinoma and normal people. The operation steps were carried out according to the manual with some modifications.
[0047] (1) Thoroughly mix 2ml of collected saliva sample with 4ml of QIAzol lysate, and place at room temperature for 5 minutes;
[0048] (2) Add 1.1ml of chloroform, mix thoroughly, and place at room temperature for 5 minutes;
[0049] (3) Under the condition of 12000g, centr...
Embodiment 2
[0068] qRT-PCR experiment of miRNA in saliva
[0069] 1. According to the miRNA microarray results, select 6 miRNAs with signal values greater than 500 in the microarray results for further experiments with qRT-PCR. The selected primers for has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730 are shown in Table 1 below. The qRT-PCR detection of miRNA was performed on the saliva of nasopharyngeal carcinoma patients and normal controls. Strict quality control was implemented throughout the study, and each sample was continuously tested three times.
[0070] Table 1
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[0072]
[0073] 2. After obtaining the informed consent of the patients, saliva samples from 80 patients diagnosed with nasopharyngeal carcinoma and 80 healthy people were collected from Jiangsu Cancer Hospital as normal controls.
[0074] 3. Use the miRNeasy Serum / Plasma Kit kit (#217184, Qiagen, Hilden, Germany) to extract RNA from the saliva of patients with ...
Embodiment 3
[0105] Embodiment 3: qPCR kit for diagnosis of human nasopharyngeal carcinoma
[0106] The above examples show that has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has-miR-4730 have positive effects on nasopharyngeal carcinoma saliva detection High diagnostic value (high sensitivity and specificity), so can be based on has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478, and has -MiR-4730 production of a kit for the diagnosis of human nasopharyngeal carcinoma. The kit includes has-miR-30b-3p primers and probes; has-miR-1202 primers and probes; has-miR-1321 primers and probes; has-miR-3612 primers and probes; 4478 primers, probes; has-miR-4730 primers, probes. Primers specifically include reverse transcription primers, quantitative PCR pre-primers and quantitative PCR post-primers, as shown in Table 1.
[0107] Of course, the kit should also include the conventional enzymes and reagents required for the corresponding PCR reactio...
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