Preparation method for serum miRNAs specific to active pulmonary tuberculosis

A pulmonary tuberculosis and active technology, applied in the field of molecular biology, can solve the problems of high cost, complicated sequencing operation, limited sequencing depth, etc., and achieve the effect of reducing the incidence rate.

Active Publication Date: 2012-10-17
ZHEJIANG UNIV
View PDF2 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the small molecular weight of miRNAs, the content in serum is small, but the quantity is huge, the traditional sequencing method is complicated to operate, expensive, limited in sequencing dept

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method for serum miRNAs specific to active pulmonary tuberculosis
  • Preparation method for serum miRNAs specific to active pulmonary tuberculosis
  • Preparation method for serum miRNAs specific to active pulmonary tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Collection and clinical testing of serum samples from patients with active pulmonary tuberculosis and healthy controls:

[0024] A total of 80 patients with active pulmonary tuberculosis and 80 healthy controls were collected. The blood samples of all participants were drawn in the morning on an empty stomach, using disposable vacuum non-anticoagulant blood collection tubes, collecting 3.0mL of peripheral blood, centrifuging within 4 hours (3000x g, 10min, 4°C), aspiration of the supernatant, and aliquoting into 200μL each Serum samples were stored in a -80°C refrigerator.

Embodiment 2

[0026] Extract total RNA from all serum samples:

[0027] Take out the serum of patients with active pulmonary tuberculosis and healthy controls at -80°C, and let it dissolve naturally at 4°C; pipette 200 μL, add it to a 1.5mL RNase-free EP tube, add 700 μL Qiagen Lysis solution, and place in a vortex Vigorously vortex and mix until fully lysed, until no white suspension can be seen; stand at room temperature for 5 minutes; add 140 μL chloroform; mix vigorously for 15 seconds, and let stand for 3 minutes; ℃; Use a pipette to transfer the supernatant (about 500 μL) to a new 1.5 mL collection tube, add 1.5 times the volume of the supernatant in absolute ethanol, mix up and down; take 700 μL of ethanol-containing lysate and add it to the column In the middle, centrifuge at 8000g for 18s, discard the lower layer, reset the collection tube on the column (repeat 2 times); add 700μL RWT solution, centrifuge at 8000g for 18s, discard the lower layer, put the column on a new collection...

Embodiment 3

[0029] Example 3: High-throughput sequencing screening technology was used to initially screen differentially expressed miRNAs in the serum of patients with active pulmonary tuberculosis and healthy controls.

[0030] 20 cases of pulmonary tuberculosis patients and 20 cases of healthy controls each provided 20mL of serum; serum requirements: after detection by Agilent Bioanalyzer, the concentration is ≥5ng / ul, and the total amount is ≥100ng; there are few impurities such as protein and salt ions, and the separation by PAGE gel electrophoresis is relatively normal ;According to the results and bioinformatics software analysis, such as normalization and differential analysis calculation methods to select differentially expressed miRNAs; through clustering methods including hierarchical clustering, Kmeans clustering and SOM, etc. to classify different samples, and differentially expressed miRNAs Similarity analysis was performed to obtain differentially expressed serum miRNAs.

...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a preparation method for serum miRNAs specific to active pulmonary tuberculosis. The method comprises the following steps: A1, collection and clinical detection of serum samples of patients with active pulmonary tuberculosis and healthy controls; A2, extraction of total RNAs from the serum samples; A3, high flux sequencing and screening of serum miRNAs specific to active pulmonary tuberculosis; and A4, designing of stem-loop reverse transcription primers, upstream primers and downstream primers of miRNAs, wherein the stem-loop reverse transcription primers are SEQ ID No. 10 to SEQ ID No. 18, a commonly-used upstream primer is SEQ ID No. 19 and a commonly-used downstream primer is SEQ ID No. 28. According to the invention, to prepare serum miRNAs specific to active pulmonary tuberculosis, high flux sequencing is used for preliminary screening of differentially expressed serum miRNAs, specific reverse transcription primers are designed by using a stem-loop method, and verification is carried out by using a fluorescence quantitative method so as to obtain the differentially expressed serum miRNAs; the serum miRNAs have remarkable significance to research on control of propagation of pulmonary tuberculosis and to reduction of the incidence rate of pulmonary tuberculosis.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for preparing active pulmonary tuberculosis specific (marker) serum miRNAs. Background technique [0002] There are 2 billion people infected with Mycobacterium tuberculosis in the world, 20 million people suffer from tuberculosis, of which 12 million cases are active tuberculosis, 8.5-9.2 million new cases of tuberculosis (pulmonary tuberculosis, TB) are added every year, and the death rate is as high as 1.2 to 1.5 million cases, which is the disease with the largest number of deaths caused by a single infection factor. Among the 22 countries with the most severe tuberculosis in the world, my country ranks second, and the epidemic situation is very severe. There are currently 4.51 million tuberculosis patients in my country, including about 1.3 million active tuberculosis patients, 1.45 million new cases per year, and a death rate of 130,000 cases per year, e...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/113C12N15/10C12Q1/68
Inventor 李继承张星
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap