Serum messenger ribonucleic acid biomarker for diagnosing recurrent spontaneous abortion, primer set and application and kit

A messenger ribonucleic acid and biomarker technology, applied in the field of serum messenger ribonucleic acid biomarkers for the diagnosis of recurrent miscarriage

Active Publication Date: 2018-09-04
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the application of mRNA in the auxiliary diagnosis of recurrent miscarriage. If the mRNA related to recurrent miscarriage can be screened out as a biomarker for early diagnosis, and the corresponding diagnostic kits can be developed, the diagnosis status of recurrent miscarriage will definitely be improved. It is a powerful impetus, and it also opens up a new way for its drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • Serum messenger ribonucleic acid biomarker for diagnosing recurrent spontaneous abortion, primer set and application and kit
  • Serum messenger ribonucleic acid biomarker for diagnosing recurrent spontaneous abortion, primer set and application and kit
  • Serum messenger ribonucleic acid biomarker for diagnosing recurrent spontaneous abortion, primer set and application and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Decidual tissue mRNA screening

[0079] 1 material

[0080] 1.1 Selection of research objects and sample collection

[0081] (1) According to the diagnostic criteria: pregnant women with 2 or more consecutive pregnancy losses before 20 weeks of pregnancy or fetal weight less than 500g are the case group; according to age, gestational weeks, BMI and other indicators, the frequency matching method is used to select children Pregnant women with normal ability and non-medical reasons for miscarriage (without signs and signs of miscarriage, no history of miscarriage, and at least one normal child born) were used as the control group.

[0082] (2) B-ultrasound shows no germ or fetal heartbeat, and abortion caused by factors such as monogenic genetic diseases, polygenic genetic diseases, chromosomal abnormalities, and gene mutations must be excluded.

[0083] (3) Grouping of research objects:

[0084] Group A: the control group of pregnant women who chose abortion ...

Embodiment 2

[0094] Example 2: Detection of the expression levels of ZBP1, ARID5A, and ALPL in decidua tissue

[0095] 1. Materials

[0096] 1.1 Primer design

[0097] The primers (Table 1) were designed for quantitative Real-time PCR detection of each mRNAs in the decidua tissues of 63 cases of unexplained recurrent abortion cases and 63 cases of non-medical reasons selected abortion control samples.

[0098] 1.2 Preparation of cDNA samples

[0099] a) Take 100 mg of decidual tissue; b) Add 900 μl Trizol, shake and mix, centrifuge at 12,000 rpm for 15 minutes at 4°C, and discard the waste liquid in the lower layer; c) Add 1.5 times the volume of supernatant, shake and mix, and transfer to centrifugation Centrifuge the column at 12,000 rpm for 15 seconds, discard the lower layer of waste liquid; d) add 700 μl of RWT buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer of waste liquid. e) Add 500 μl of RPE buffer to the spin column, centrifuge a...

Embodiment 3

[0106] Embodiment 3: qRT-PCR method detects the expression level of ZBP1, ARID5A, ALPL in serum

[0107] 1 material

[0108] 1.1 Selection of research objects and sample collection

[0109] The selection of research objects and sample collection in this embodiment are the same as those in Embodiment 1.

[0110] 1.2 Reagents

[0111] The reagents used in qPCR are the same as in Example 1.

[0112] 2 methods

[0113] 2.1 Extraction of total RNA

[0114] Serum samples from 63 cases of normal control group and 63 cases of recurrent miscarriage case group were taken as subjects, and 5ml of fresh heparin anticoagulated blood was centrifuged in a centrifuge at 3000rpm for 5min, and the supernatant was divided into clean 1.5ml EP tubes, 100μl per tube . Add 900 μl TRIzol to the EP tube, shake and mix thoroughly, centrifuge at 12,000 rpm for 15 min at 4°C, and immediately transfer the supernatant to a clean 1.5ml EP tube. Add 1.5 times the volume of supernatant water phase absol...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a serum messenger ribonucleic acid biomarker for diagnosing recurrent spontaneous abortion, a primer set and application and akit. The serum messenger ribonucleic acid biomarker comprises at least one of first mRNA with 90% or more of homology with a nucleotide sequence shown by a SEQ ID NO: 1, second mRNA with 90% or moreof homology with a nucleotide sequence shown by a SEQ ID NO: 2 and third mRNA with 90% or more of homology with a nucleotide sequence shown by a SEQ ID NO: 3. Serum mRNA is a novel biomarker which isstable, minimally invasive, easy to detect and quantitatively accurate, and can greatly improve the sensitivity and specificity of recurrent spontaneous abortion diagnosis.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a serum messenger ribonucleic acid biomarker, primer set, application and kit for diagnosing recurrent miscarriage. Background technique [0002] Spontaneous abortion is one of the most common diseases in women of childbearing age. Among women of childbearing age, 15-20% of clinical pregnancies end in spontaneous abortion. According to epidemiological investigation and research, spontaneous abortion is a multi-cause disease. The main cause of spontaneous abortion that has been identified so far is still the abnormality of embryonic chromosomes. Abnormal anatomy of the maternal reproductive tract, endocrine abnormalities, immune disorders, abnormal blood coagulation, abnormal metabolism, etc. may also lead to miscarriage. However, even after detailed and strict etiological screening, 40% to 60% of spontaneous abortions still have unknown etiology, which limits ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/156
Inventor 王嵘于明明徐俏俏杜桂珍韩莉陆春城夏彦恺王心如
Owner NANJING MEDICAL UNIV
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