Osteosarcoma metastasis inhibitor composition and application thereof
An osteosarcoma and reagent technology, applied in the field of molecular biology, can solve the problem of lack of effective targets for osteosarcoma diagnosis and treatment
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0057] The collection of embodiment 1 sample and total RNA extraction
[0058] 5 patients with metastatic osteosarcoma and 5 healthy controls. Five patients in the metastatic osteosarcoma group and five in the control group were required to fast for at least 12 hours, and at room temperature from 7:00 to 8:00 the next morning, 10ml of venous blood was drawn into ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and extracted Peripheral blood mononuclear cell PBMCs, add 1ml Trizol reagent (Invitrogen Company), mix thoroughly, and store the specimen at -80°C for RNA extraction. All blood samples and pathological results should be authentic and reliable, the study was approved by the ethics committee, and the patients gave informed consent.
[0059] RNA extraction standards: RNA purity: OD260 / 280≧1.8, 28S / 18S≧1; RNA integrity: RIN value≧7.0. RNA integrity detection method: Agilent2100 (RNA6000Nanokit), agarose gel electrophoresis (agarose gel concentration: 1% agarose...
Embodiment 2
[0060] Example 2 Sequencing and Data Analysis
[0061] Sequencing: miRNA was sequenced using lluminaHiseq2500 / Miseq second-generation high-throughput sequencing technology, and data processing was completed by removing joints, removing low-quality, and decontaminating processes to obtain final data.
[0062]The miRNA raw data were background-corrected by transcriptome data analysis software, and then the t-test was performed to obtain the P value, and then the Fisher test was used to combine the P values to screen for differentially expressed miRNAs. Setting p value <0.01, 12 differentially expressed miRNAs were screened out, including 11 miRNAs whose expression levels were down-regulated. Among them, miR-6884-5p, which down-regulated the expression significantly, entered our research scope.
Embodiment 3
[0063] Example 3 Real-timePCR detection of miR-6884-5p gene expression in osteosarcoma tissue
[0064] 1 sample collection:
[0065] The peripheral blood of 49 patients with metastatic osteosarcoma tumors and 63 healthy controls came from the hospital (collection time from April 2014 to August 2015).
[0066] 2 total RNA extraction:
[0067] Removal of RNase for related experimental items:
[0068] ① Rinse and soak all glassware with DEPC before use, 120°C for 20 minutes under high pressure, and 180°C for more than 2 hours.
[0069] ②Plastic utensils (such as: EP tubes / tips) need to be soaked in 0.1% DEPC water overnight before use, and then the liquid is controlled to dry, 120°C and high pressure for 20 minutes, and dried in an oven for later use.
[0070] leukapheresis
[0071] (1) Take 2m1 anticoagulated peripheral blood (blood collection time does not exceed 3h);
[0072] (2) Add an equal volume of sterile PBS to the peripheral blood and mix thoroughly to form a cell ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More