miRNAs as targets for diagnosis and treatment of osteosarcoma
A technology for osteosarcoma and a diagnostic tool, which is applied in the field of diagnosis and treatment of osteosarcoma by miR-4714-3p, can solve problems such as abnormal gene expression and induced diseases, and achieve the effect of small diagnosis in time, reduce mortality, and realize early diagnosis
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Embodiment 1
[0037] Example 1 High-throughput sequencing screening of miRNAs associated with osteosarcoma
[0038] 1. Sample collection
[0039] 5 patients with primary osteosarcoma and 5 normal people. Subjects were required to fast for at least 12 hours, and at room temperature from 7:00 to 8:00 in the morning of the next day, 10ml of venous blood was drawn into EDTA anticoagulant tubes to extract PBMCs from peripheral blood mononuclear cells, and 1ml of Trizol was added Reagents (Invitrogen Company), mixed thoroughly, and samples were stored 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.
[0040] 2. RNA extraction from blood monocytes
[0041] Thaw the cells in step (1), add about 1 / 5 of the volume of chloroform, mix well by inverting up and down for about 1 minute, and let stand at room temperature for 5 minutes. After centrifugation at ...
Embodiment 2
[0052] Example 2 QPCR verification of differentially expressed miR-4714-3p
[0053] 1. According to the high-throughput sequencing results in Example 1, miR-4714-3p was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 50 cases of normal people and 50 cases of osteosarcoma patients were selected to separate and store blood mononuclear cells.
[0054] 2. The RNA extraction process is the same as in Example 1.
[0055] 3. Reverse transcription: mix 10pg-1μg total RNA template with 2μl 10* buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl ribonuclease (RNase) inhibitor and ribonuclease free water (RNase free water ) were mixed, the final volume was 20 μl, and incubated at 37° C. for 1 h. Then add 1 μl 0.5 μg / μl Oligo(dT) specific RT primer to the reaction tube, incubate at 70°C for 5 minutes and immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers. Finally, mix the above 2...
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