Application and kit of miRNA markers in the preparation of products for the evaluation of the efficacy of olanzapine in the treatment of schizophrenia
A technology for schizophrenia and efficacy evaluation, applied in biochemical equipment and methods, and microbial determination/examination, etc., can solve problems such as adverse reactions and rebound of positive symptoms
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Embodiment 1
[0056] Example 1: High-throughput sequencing screening of miRNA markers related to the efficacy of olanzapine in the treatment of schizophrenia
[0057] 1. Screening of miRNA markers in peripheral blood related to the efficacy of olanzapine in the treatment of schizophrenia
[0058] 1.1. Serum sample processing, blood collection, centrifugation, and storage for later use
[0059] Peripheral blood of 10 schizophrenic patients who failed olanzapine treatment and 10 schizophrenic patients who were effective olanzapine treatment were collected. The patients all gave informed consent, and all the above specimens were obtained with the approval of the organizational ethics committee. All included subjects took 3ml of fasting venous blood in the morning, and immediately separated the serum by centrifugation at 2000rpm for 8min in a horizontal centrifuge, put the aspirated supernatant in a centrifuge tube, and centrifuged at 14000rpm for 10min at 4°C, and put the aspirated supernatant...
Embodiment 2
[0079] Example 2: qRT-PCR verification of expression differences of miR-143-3p
[0080] 2.1. Serum sample processing, blood collection, centrifugation, and storage for later use
[0081] Among the patients in Example 1, 4 cases of peripheral blood were collected from patients with schizophrenia who were ineffective in the treatment of olanzapine and 4 cases in each of patients with schizophrenia who were effective in the treatment of olanzapine, and the rest of the operations were the same as in Example 1;
[0082] 2.2. Extraction of total RNA
[0083] Total RNA extraction steps are the same as in Example 1;
[0084] 2.3. Use ultra-micro spectrophotometer to detect the quality of RNA
[0085] Take 2 ul of dissolved RNA, and measure the absorbance at 260 mm with an ultramicro spectrophotometer to calculate the yield of RNA.
[0086] 2.4. Perform reverse transcription to obtain cDNA, and perform fluorescence quantitative detection on the obtained cDNA;
[0087] The primers o...
Embodiment 3
[0107] Example 3: Research on the differential expression of miR-143-3p gene in other neural cell lines
[0108] 3.1. Cell culture
[0109] Inoculate human neuroblastoma cells (SH-SY5Y cells) in 6-well cell culture plates, add MEM:F12 medium containing 10% fetal bovine serum, and place in an incubator at 37°C, 5% CO2 and saturated humidity In , cells in the logarithmic growth phase were used for the experiment. When the degree of confluence is about 80%. SH-SY5Y cells were treated with olanzapine at concentrations of 0 μM (control group), 1 μM, and 10 μM for 24 hours.
[0110] 3.2. Extraction of RNA
[0111] (1) Aspirate the medium in the well plate, wash it twice with PBS, add 1mL Trizol to each well and pipette repeatedly for 10-15 times, then let stand at room temperature for 5 minutes;
[0112] (2) Collect the cell suspension into a 1.5mL enzyme-free EP tube, vortex for 10s, and then let it stand on ice for 5min; add 200μL of pre-cooled chloroform to each tube, shake i...
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