Renal cell carcinoma miRNA molecular marker and application thereof
A technology for molecular markers and renal cell carcinoma, applied in the field of miRNA molecular markers for renal cell carcinoma
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Embodiment 1
[0040] Embodiment 1. prepares the test kit (50 times of reaction) of predicting RCC patient's transfer and prognosis
[0041] 1. Trizol: 50ml;
[0042] 2. Chloroform: 20ml;
[0043] 3. Isopropanol: 30ml;
[0044] 4.75% ethanol; 60ml;
[0045] 5. DEPC water: 10ml;
[0046] 6. miRNA reverse transcription specific primer (its sequence is shown in SEQ NO: 1-4): 100ul;
[0047] 7. 200U / ul M-MLV reverse transcriptase: 50ul;
[0048] 8. SYBR qPCR Mix: 500ul;
[0049] 9. 10uM target gene-specific primer (its sequence is shown in SEQ NO:5-10): each 100ul;
[0050] 10. 10uM internal reference gene U6-specific primer (its sequence is shown in SEQ NO: 11-12): 100ul each.
Embodiment 2
[0051] Example 2. Detection and analysis of miRNA molecular markers in tissue samples
[0052] 1. Collect the RCC tissues to be tested, normal paracancerous tissues, and distantly metastatic RCC tissues, wash them with normal saline, and freeze them in liquid nitrogen.
[0053] 2. Tissue RNA extraction: Add liquid nitrogen into the mortar, cut the tissue into pieces and grind it in liquid nitrogen, take 100mg of tissue and add it to 1ml Trizol, and mix well. Leave it at room temperature for 5 minutes, add 200uL / tube of chloroform, shake vigorously and mix for 15 seconds. Centrifuge at 12,000 rpm for 15 minutes. Carefully pipette the upper aqueous phase into a new Ep tube, add an equal volume of isopropanol, invert and mix well, place at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes. Carefully discard the supernatant, add 75% ethanol, mix well and centrifuge at 8000 rpm for 8 minutes. Remove the supernatant, and add DEPC water to dissolve the RN...
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