Enhancer RNA combination used for diagnosing unexplained recurrent spontaneous abortion, primer group and application and kit thereof

A technology for recurrent miscarriage and unclear diagnosis, applied in the field of genetic engineering, to achieve the effects of wide detection spectrum, high sensitivity and low cost

Active Publication Date: 2017-12-08
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 enhancer RNA in the auxiliary diagnosis of unexplained recurrent miscarriage. If the enhancer RNA related to unexplained recurrent miscarriage can be screened out as a biomarker, and the corresponding

Method used

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  • Enhancer RNA combination used for diagnosing unexplained recurrent spontaneous abortion, primer group and application and kit thereof
  • Enhancer RNA combination used for diagnosing unexplained recurrent spontaneous abortion, primer group and application and kit thereof
  • Enhancer RNA combination used for diagnosing unexplained recurrent spontaneous abortion, primer group and application and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Screening, Screening and Confirmation of Enhancer RNA in Unexplained Recurrent Miscarriage

[0041] 1. Materials

[0042] 1.1 Organization

[0043] The clinical tissue samples of the present invention are all from patients with unexplained recurrent abortion diagnosed in Huai'an First People's Hospital Affiliated to Nanjing Medical University from March 2015 to July 2016. Exact match. The use of all specimens was signed by the patients or their trustees, and the standards of the Medical Ethics Committee of Nanjing Medical University were obtained.

[0044] 1.2 Reagents

[0045] TRIzol TM Reagent was purchased from Invitrogen, USA. The reverse transcription kit (DRR036A) was purchased from Takara, Japan. The SYBR reagent used in real-time quantitative PCT was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd. PCR primers were designed and synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Transcriptome sequencing and data analysis were comp...

Embodiment 2

[0052] Example 2: qRT-PCR method to verify transcriptome sequencing results

[0053] 1. Materials

[0054] 1.1 Organization

[0055] The clinical tissue samples of the present invention are the same as in Example 1.

[0056] 1.2 Reagents

[0057] The reagents used in qPCR are the same as in Example 1.

[0058] 2. Method

[0059] 2.1 Preparation of cDNA samples

[0060] Grind the tissue in liquid nitrogen, add 1 mL TRIzol for every 50-100 mg tissue, and use a homogenizer for homogenization. The sample volume should not exceed 10% of the volume of TRIzol. Place the homogenized sample at room temperature (15-30°C) for 5 minutes to completely separate the nucleic acid-protein complex. Add 0.2 mL of chloroform for every 1 mL of TRIzol used, shake vigorously for 15 s, and place at room temperature for 3 min. Centrifuge at 10 000 g for 15 min at 4°C. The sample is divided into 3 layers: the bottom layer is a red organic phase, the upper layer is a colorless aqueous phase and...

Embodiment 3

[0071] Example 3: qRT-PCR method to detect the expression of enhancer RNA lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 in serum

[0072] 1. Materials

[0073] 1.1 Serum

[0074] The collection of clinical serum samples of the present invention is the same as in Example 1.

[0075] 1.2 Reagents

[0076] The reagents used in qPCR are the same as in Example 1.

[0077] 2. Method

[0078] Add 1 mL TRIzol to 200 μl serum sample, mix thoroughly, and let stand at 4°C for 5 minutes to completely separate the nucleic acid-protein complex. Subsequent methods are the same as in Example 1 2.2.

[0079] 3. Results

[0080]Compared with normal pregnant women, lnc-ERGIC1-4, lnc-PBK-2, and lnc-SLC4A1-1 were all highly expressed in the serum of recurrent patients (such as image 3 shown), suggesting that this group of enhancer RNAs is associated with unexplained recurrent miscarriage and can be used for the diagnosis of the disease.

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Abstract

The invention belongs to the field of genetic engineering and relates to an enhancer RNA combination used for diagnosing unexplained recurrent spontaneous abortion, a primer group and an application and a kit thereof. The enhancer RNA combination comprises a first enhancer RNA with more than 90% of homology with a nucleotide sequence shown as SEQ ID NO:1, a second enhancer RNA with more than 90% of homology with a nucleotide sequence shown as SEQ ID NO: 2, and a third enhancer RNA with more than 90% of homology with a nucleotide sequence shown as SEQ ID NO: 3. The screened enhancer RNA combination in the invention serves as a novel marker, the kit used for diagnosing or monitoring the unexplained recurrent spontaneous abortion is prepared, and the kit has the advantages of high sensitivity, wide detection spectrum, low cost and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to an enhancer RNA combination and a primer set for diagnosing unexplained recurrent miscarriage, and an application and a kit of the enhancer RNA combination and the primer set. Background technique [0002] Recurrent spontaneous abortion (RSA) refers to clinically two or more consecutive pregnancy losses before 20W or fetal weight less than 500g, and its incidence rate accounts for about 1% to 3% of pregnancy outcomes. The etiology of RSA is extremely complex, and the etiology that has been identified so far includes chromosomal abnormalities, abnormal reproductive anatomical structures, endocrine disorders, reproductive system infections, autoimmunity, and environmental factors, but there are still 50% to 60% of RSA cases that cannot be identified clinically. It is called unexplained recurrent spontaneous abortion (URSA). Abnormal embryonic development is the m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/178
Inventor 陆春城黄振遥范赟韩秀梅黄晓敏韩莉夏彦恺王心如
Owner NANJING MEDICAL UNIV
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