Diagnostic markers for endometrial cancer
A technology of endometrial cancer and drugs, which is applied in the field of markers for the diagnosis and treatment of endometrial cancer, and can solve the problems of less research on differential expression of miRNA
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Embodiment 1
[0060] Example 1 Screening of miRNAs associated with endometrial cancer
[0061] 1. Sample acquisition: 10 cases of normal endometrial tissue and endometrial cancer tissue were collected respectively. All the above specimens were obtained with the consent of the organizational ethics committee.
[0062] 2. Extraction of total RNA from samples
[0063] Total RNA in advance using QIAGEN Tissue RNA Extraction Kit. Specific steps are as follows:
[0064] 1) In a clean area with less RNase interference, use a mortar containing an appropriate amount of liquid nitrogen to weigh about 20 mg of the tissue sample, and grind it to powder with a pestle;
[0065] 2) Transfer the sample to a 2ml centrifuge tube without RNase;
[0066] 3) Add 300 μl Lysis solution, place in a homogenizer, and grind thoroughly for 1-5 minutes;
[0067] 4) 12000g, 4°C, centrifuge for 10min, transfer the supernatant to a new 1.5ml centrifuge tube;
[0068] 5) Add 600μl RNase-Free Water and mix with a vort...
Embodiment 2
[0091] Example 2 QPCR verification of differentially expressed miRNA-3170
[0092] 1. According to the detection results of the miRNA chip, select miRNA-3170 for large-sample QPCR verification. According to the sample collection method in Example 1, 60 endometrial cancer tissue samples and 60 normal endometrial tissue samples were selected.
[0093] 2. The RNA extraction process is the same as in Example 1.
[0094] 3. Reverse transcription:
[0095]1) Mix 10pg-1μg total RNA template with 2μl 10× buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl RNase inhibitor and RNase free water , the final volume was 20 μl, and incubated at 37°C for 1h.
[0096] 2) Add 1 μl of 0.5 μg / μl Oligo(dT) specific RT primer to the reaction tube, and incubate at 70° C. for 5 minutes.
[0097] 3) Immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers.
[0098] 4) Mix the above 20 μl reaction mixture with 4 μl 5× buffer, 1 μl dNTP (10 mM), 0.5 ...
Embodiment 3
[0114] Example 3 Detection of miRNA-3170 expression in endometrial cancer cells by QPCR
[0115] 1. Amplification and identification of miRNA-3170 plasmid
[0116] 1) According to the sequence information of miRNA-3170, Dalian Bao Biotechnology Co., Ltd. designed and synthesized miRNA-3170 inhibitor plasmid and negative control plasmid of random control sequence.
[0117] 2) Plasmid transformation into DH5α competent strain
[0118] ①Take out 100 μl of DH5α competent bacteria from the -80°C refrigerator and melt on ice;
[0119] ② Add 10 μl pMKITeno plasmid to 100 μl DH5α competent bacterial solution, flick and rotate the bottom of the tube to mix well, and place in ice bath for 30 minutes;
[0120] ③ Place in a water bath at 42°C for 90 seconds after heat shock; immediately place in an ice bath for 90 seconds;
[0121] ④ Add 800 μl of Amp-LB culture solution, gently blow and mix, and incubate at 37°C on a constant temperature shaker at 100 rpm for 1 hour to recover the bac...
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