Recurrent spontaneous abortion relevant microRNA and applications thereof
A technology for repeated spontaneous abortions and uses, applied in the fields of biotechnology and medicine, can solve the problems of RM identification and prediction methods that are poorly understood
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[0088] The preparation of the miRNA chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The miRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and then fixing it by standing overnight. If the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-Radioactive Operation Manual" edited by Wang Shenwu; J.L.erisi, V.R.Iyer, P.O.BROWN. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 1997; 278:680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.
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Embodiment 1
[0107] Example 1 Screening of Serum Markers
[0108] method:
[0109] Patients with recurrent miscarriage who visited the family planning clinic of the hospital were selected, and research subjects were recruited according to the following inclusion criteria. After informed consent of the research subjects, 15 cases of recurrent miscarriage were collected.
[0110]
[0111] 1. Real-time fluorescent quantitative PCR detection of expression level of hsa-miR-3074-5p
[0112] 1) Experimental reagents and their preparation
[0113] (1) RT primers: provided by Ribobio (RN: R10031.2), operated according to the instructions, the lyophilized powder was mixed with RNase-free water to prepare a 5 μM primer storage solution, and then stored in aliquots; before use , diluted 10 times (45 μl water + 5 μl storage solution), and prepared as 500 nM RT primer working solution.
[0114] (2) RT enzyme: MulV Reverse Transcriptase, a product of Roche Company.
[0115] (3) 10×PCR Buffer: a pr...
Embodiment 2
[0154] Embodiment 2 prepares miRNA chip
[0155] The miRNA sequence (SEQ ID NO: 1) provided by the present invention is converted into a complementary sequence, and a 10-20nt connection sequence is added to both ends of the sequence according to the GC ratio and other characteristics of the generated sequence; the core sequence is different, and the connection sequence is also different. The junction sequence can be randomly generated by the program, and the probes formed by the junction sequence and the core sequence meet the following conditions:
[0156] 1) In the probe sequence, the number of the same nucleotide (A, C, G, T) cannot exceed 50% of the total number of sequences;
[0157] 2) The number of any continuous A, T or C, G cannot exceed 25% of the total number of sequences;
[0158] 3) G and C content account for 40%-60% of the total number of sequences;
[0159] 4) The probe sequence cannot self-hybridize, that is, the length of the complementary segment in the pr...
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