Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers

A preeclampsia and peripheral blood technology, applied in the fields of genetic engineering and clinical medicine, can solve the problems of limitations of research methods, poor research repeatability, failure to obtain clinically meaningful results, etc., and achieve the effect of optimal cluster density

Inactive Publication Date: 2014-08-20
SHANXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the small sample size, the reproducibility of the study was poor, and due to t

Method used

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  • Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers
  • Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers
  • Peripheral white blood cell miRNA markers associated with onset of human preeclampsia and application of miRNA markers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Research object selection and group basis

[0059] Pregnant women who gave birth in the Obstetrics and Gynecology Department of the Second Hospital of Shanxi Medical University from April 2013 to January 2014 were selected as the research subjects.

[0060] Group A: experimental group, 5 patients with preeclampsia, no other major systemic diseases; Group B: healthy control group: 5 people, no other major systemic diseases; analysis between groups was done after the sequencing of the two groups was completed.

[0061] Group 1: 13 normal age preeclampsia patients (PE) group, with an average age of 25.69±1.32 years; Group 2: 13 elderly preeclampsia patients (PA) group, with an average age of 34.84±3.18 years; Group 3: Preeclampsia with complications (PC) group of 13 people, with an average age of 30.08±2.65 years old; Group 4: normal pregnant women at the same period (N) control group with 13 people, with an average age of 29.07±2.59 years old. The total RNA...

Embodiment 2

[0062] Example 2: RNA sequencing of peripheral blood leukocytes using miRNA high-throughput HiSeq2000 and Genbank database comparison results

[0063] The total RNA of the extracted peripheral blood leukocytes was taken and dissolved in DEPC-treated water (Ambion) at a concentration of 25-500 ng / ul to prepare an RNA library; cDNA samples were obtained by RNA reverse transcription.

[0064] 1. Reverse transcription reaction system and conditions: first add 0.5 μl RT-Primer (100 μM), place at 65°C for 10 minutes, centrifuge and cool to room temperature, then add in sequence: ① 5x first strand buffer 2.0 μl; ② 10mM dNTP 0.6 μl; ③ l00mM DTT 1 μl; ④ Rnase OUT (40U / μl) lμl, mix well, centrifuge at 42°C for 3 minutes, finally add lμl Superscript (200U / μl), the total volume is 20μl, mix well, react at 42°C for 1 hour and then denature at 7°C 15 minutes.

[0065] 2. The RT-PCR amplification reaction system is shown in Table 1, and the RT-PCR amplification reaction conditions are shown...

Embodiment 3

[0087] Example 3: Real-time quantitative PCR verification of differential expression of miRNA in peripheral blood leukocytes of patients with preeclampsia

[0088] Use the miRcute miRNA First-Strand cDNA Synthesis Kit kit and PCR instrument from TIANGEN Company, and operate according to the instructions attached to the kit:

[0089] 1. Add the following reagents to the RNase Free reaction tube pre-cooled on ice to a total volume of 20ul (E.coli Poly(A) Polymerase is added at the end), as shown in Table 5.

[0090] Table 5: Poly (A) treatment at the 3' end of miRNA

[0091]

[0092] 2. Gently mix the reaction solution prepared above with a pipette, centrifuge briefly and react at 37°C for 60 minutes; prepare the reaction solution on ice for reverse transcription reaction, see Table 6 for the reverse transcription reaction system:

[0093] Table 6: Poly (A) modified miRNA for reverse transcription reaction

[0094]

[0095] 3. Gently mix the prepared reaction solution wi...

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Abstract

The invention belongs to the field of genetic engineering and clinical medicine. The invention aims to provide peripheral white blood cell miRNA markers associated with the onset of human preeclampsia. The invention also aims to provide an application of the miRNA markers. The miRNA markers can be many of has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p. The peripheral white blood cell miRNA markers include has-miR-15a-3p, has-miR-31-3p, has-miR-451a and has-miR-122-5p. The peripheral white blood cell miRNA marker is applied to preparation of reagent for diagnosis or monitoring of human preeclampsia. The reagent is capable of determining the expression of the peripheral white blood cell microRNA marker in peripheral white blood cell. According to the invention, verifications are carried out on samples in line with preeclampsia and samples of a healthy control population to prove that the expressions of the markers are significantly different and the markers have stability and thus the results demonstrate that the markers have specificity and can be used as markers.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and clinical medicine, and specifically relates to peripheral blood leukocyte miRNA markers related to the occurrence of human preeclampsia and applications thereof. Background technique [0002] Preeclampsia (Preeclampsia, PE) is a common idiopathic multi-organ damage disease during pregnancy, which is characterized by hypertension, proteinuria and edema after 20 weeks of gestation, which may lead to placental abruption, DIC, uterine The occurrence of serious complications such as epilepsy, HELLP syndrome, renal failure, cerebrovascular accident and heart failure. The incidence of PE during pregnancy is 2% to 8% worldwide, and it is currently the main cause of maternal, fetal and infant morbidity and mortality. The clinical manifestations of PE have obvious heterogeneity, which makes it more difficult to obtain effective clinical treatment. [0003] At present, there are two main theories to...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/68
Inventor 王永红郝敏杨元元王文君
Owner SHANXI MEDICAL UNIV
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