Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof

A marker, diabetes technology, applied in the field of genetic engineering and reproductive medicine

Inactive Publication Date: 2011-04-27
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, there are currently no reports of relatively stable biomarkers for GDM-assisted early diagnosis. If GDM-specific or abnormally expressed serum/plasma miRNAs

Method used

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  • Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof
  • Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof
  • Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof

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Embodiment 1

[0069] The collection of embodiment 1 sample and the arrangement of sample data

[0070] The inventor has collected a large number of peripheral blood samples of pregnant women at 16-19 weeks of pregnancy from Nanjing Maternal and Child Health Hospital since 2008 (the samples used for research were collected at the same period, and the sampling, packaging, and storage conditions were uniform). In order to sort out the data, the inventor selected 120 samples that meet the following criteria as experimental samples for TLDA chip detection and a series of subsequent qRT-PCR verifications:

[0071] 1. Healthy pregnant women at 16-19 weeks of gestation at the time of blood collection

[0072] 2. Pregnant women who were diagnosed as GDM by OGTT during GDM screening at 24-28 weeks of gestation were defined as cases

[0073] 3. The above-mentioned research subjects did not develop GDM during GDM screening at 24-28 weeks of gestation, and healthy pregnant women who matched the case ag...

Embodiment 2

[0075] TLDA chip detection of miRNA in embodiment 2 serum / plasma

[0076]The above-mentioned 24 eligible GDM cases and 24 healthy controls were detected by TLDA chip to obtain relevant results. The specific steps are:

[0077] 1. Take 600 μl of serum from the "gestational diabetes case" group and the "healthy female control" group, and add 3 times the volume of Trizol reagent;

[0078] 2. Phase separation: place at room temperature for 15 minutes, add final concentration of 10 -4 Pmol / μl of cel-39 (TAKARA) was used as an internal reference, then chloroform equal to the volume of plasma was added, shaken for 50 s, room temperature for 15 min, 14,000 rpm, 4°C, and centrifuged for 15 min;

[0079] 3. RNA precipitation: transfer the water phase to a new 15ml centrifuge tube, add 1.5 times the volume of the water phase in absolute ethanol, and mix well;

[0080] 4. Enrich RNA with the QIAGEN miRNeasy kit kit: pipette 700 μl of sample into the spin column each time, centrifuge at...

Embodiment 3

[0086] qRT-PCR experiment of miRNA in embodiment 3 serum / plasma

[0087] According to the above TLDA results, miR-1, miR-125b, miR-132, miR-29a, miR-203, miR-222, miR-378, miR-518d-3p, miR-632, miR-923, miR- Primers for reverse transcription and qRT-PCR were designed for 11 miRNAs such as 99a. The qRT-PCR detection of miRNA was performed on the serum individual of the "GDM case" group and the "healthy control" group, as shown in Table 1. Strict quality control was implemented throughout the study. Each sample was tested three times consecutively. All assays were blinded, that is, performed without knowledge of the sample background to avoid bias. The dye method and the probe method were used for qRT-PCR detection respectively.

[0088] (1) Preparation of RNA samples: a) Take 100 μl of serum; b) Add 3 times the volume of Trizol and leave it at room temperature for 15 minutes, adding a final concentration of 10 -4 pmol / μl cel-39 (TAKARA) was used as an internal reference, t...

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Abstract

The invention belongs to the fields of genetic engineering and reproductive medicine and relates to a serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application of the serum/plasma miRNA marker. The marker is a combination of miR-132, miR-29a and miR-222. The marker and a prime thereof can be used for preparing a diagnosis kit and used for auxiliary early-stage diagnosis of the gestational diabetes.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a serum / plasma miRNA marker related to gestational diabetes and its application. Background technique [0002] Gestational diabetes mellitus (GDM) refers to varying degrees of abnormal glucose tolerance that occurs or is discovered for the first time during pregnancy. If it occurs in the first trimester, the possibility of abnormal glucose tolerance already existing before pregnancy cannot be ruled out. GDM is one of the most common complications during pregnancy. About 3%-8% of pregnant women develop GDM during pregnancy. It is particularly noteworthy that the incidence of GDM continues to rise with the increase in the obesity rate of women of childbearing age. [0003] GDM can bring a series of complications to the mother and fetus, such as increasing the incidence of spontaneous abortion, pregnancy-induced hypertension syndrome, polyhydramnios, et...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12Q1/68
Inventor 沙家豪胡志斌赵纯潘世扬董静石中华霍然
Owner NANJING MEDICAL UNIV
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