Endometrial cancer biological marker
An endometrial cancer, substance technology, applied in the field of treatment, prediction of prognosis, cancer diagnosis, which can solve the problems of elevated levels, lack of tissue sensitivity and specificity, etc.
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Embodiment 1
[0069] Example 1 Gene Chip Screening for Differentially Expressed Genes
[0070] 1. Sample collection:
[0071] Endometrial cancer tissue samples: Patients with endometrial cancer were collected, all of whom underwent surgical treatment, and 10 surgical paraffin specimens were collected. All patients were diagnosed with endometrial cancer by pathological examination. Other enrollment conditions are: all patients have not received any treatment before admission; no other malignant tumors; no other hormone-related diseases; complete clinical data.
[0072] 10 cases of endometrial cancer patients, the average age of onset was 58 years old. The main clinical manifestations of patients are irregular vaginal bleeding, lower abdominal pain, menstrual disorders, vaginal discharge, etc. There are also some patients who have no obvious symptoms and are found in physical examination. The endometrial cancer specimens were sectioned by HE staining, and the histomorphological diagnosis w...
Embodiment 2
[0084] Example 2 Large sample verification screened out differentially expressed genes
[0085] Considering the gene that has not been studied in the prior art on the correlation between this gene and endometrial cancer as a candidate gene, and considering the results of gene sequencing, select the SPARCL1 gene (its expression is down-regulated in endometrial cancer tissue) for verification .
[0086] 1. Sample collection
[0087] According to the method of Example 1, 50 cases of endometrial cancer tissues and 60 cases of normal endometrial tissues were collected.
[0088] 2. Validation at the mRNA level
[0089] 2.1 Extract tissue RNA
[0090] Step is with embodiment 1.
[0091] 2.2 Reverse transcription
[0092] Using a reverse transcription kit, 1 μg of total RNA was reverse-transcribed with reverse transcription buffer to synthesize cDNA. Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, and add the following components in PCR tubes: DEPC ...
Embodiment 3
[0116] Example 3 Overexpression of SPARCL1 gene
[0117] 1. Plasmid construction
[0118] Amplification primers are designed according to the coding sequence of the SPARCL1 gene, and the design of primers is well known to those skilled in the art. Amplify the coding sequence of the full-length SPARCL1 gene from the cDNA library of adult fetal brain (clontech company, article number: 638831), insert the above cDNA sequence into the eukaryotic cell expression vector pcDNA3.1, and connect the obtained recombinant vector pcDNA3.1 -SPARCL1 was used in subsequent experiments.
[0119] 2. Culture and transfection of endometrial cancer cells
[0120] 2.1 Cell culture
[0121] The Ishikawa3-H-12 cell line was cultured in RPMI 1640 medium (containing 10% fetal bovine serum, 100 U / ml penicillin, 100 g / ml streptomycin) in 50 ml culture flasks, at 37 °C, 5% CO 2Continuous cultivation in a humidified incubator.
[0122] 2.2 Cell transfection
[0123] 1. The day before transfection, 0....
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