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Small RNA and application thereof

A technology of tiny nucleic acids and oligonucleotides, applied in the field of biomedical materials, can solve the problems of lack of diagnostic sensitivity and repeatability of patients, and achieve the effect of convenient sampling

Active Publication Date: 2010-03-31
SUZHOU GENEPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] It can be seen from the above two criteria that they both lack the sensitivity and reproducibility of the patient's diagnosis
Also, there are currently no quantitative criteria for diagnosing AS

Method used

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  • Small RNA and application thereof
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  • Small RNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation of embodiment 1 total RNA

[0041] A: Isolation of peripheral blood mononuclear cells (PBMCs)

[0042] (1). Collect blood with an anticoagulant tube (containing 0.2ml of anticoagulant solution), shake well, and add Hank's solution or PBS equal volume (1:1) to dilute the blood.

[0043] (2). Take 2ml of lymphocyte stratification solution and put it into a 15ml centrifuge tube.

[0044] (3). Tilt the centrifuge tube at an angle of 45°, absorb the diluted blood with a straw, and add it slowly along the wall of the test tube at a distance of 1cm from the layered liquid surface, so that the diluted blood is stacked on the layered liquid, and the interface between the two is kept clear. . (The volume ratio of diluted blood to layered fluid is about 2:1).

[0045] (4). Centrifuge in a horizontal centrifuge at 2000rpm for 20min at 18-20°C. After centrifugation, the contents are divided into four layers. The upper layer is plasma (containing platelets), the mi...

Embodiment 2

[0059] Example 2 Gene chip analysis microRNA expression profile (gene chip purchased from Invitrogen)

[0060] (1). Extraction of peripheral blood mononuclear cells, total RNA and miRNA

[0061] Extract 10 specimens of active AS patients before treatment, 10 specimens of AS patients after 12 weeks of Enbrel treatment, and 10 specimens of healthy controls with 4ml of venous blood, and extract PBMCs by the method described in Example 1. Total RNA was extracted with TRIzolReagent (Life Technologies, Inc.), and miRNA was isolated with mirVana microRNAIsolation Kit (Ambion, Inc.), and stored at -20°C.

[0062] (2). miRNA chip labeling and purification

[0063]Vacuum-dry 2--5 μg of microRNA. After drying, resuspend the microRNA sample with 4 μl Nuclease-freeWater; use mirVana TM microRNA Labeling Kit (Ambion Inc.) with Monoreactive Cy3 dye / Monoreactive Cy5dye (Amersham Pharmacia Biotech, Ltd) as fluorescent label , QIAgene PCR Purification kit (QIAGEN, Inc.); the next step of puri...

Embodiment 3

[0068] Preparation of embodiment 3cDNA (total RNA reverse transcription):

[0069] The total RNA extracted in Example 1 was reverse-transcribed to prepare a cDNA template (reverse transcription primer sequences are shown in Table 1).

[0070] A: Reverse transcription reaction system:

[0071] Reagent name (both buffer and enzyme are

promega company)

Dosage / tube

5×Reverse Transcription buffer

(buffer)

4ul

RT Primer Mix (1uM) (primer mixture)

1.25ul

dNTP (10mM) (four deoxyribonucleotides

Mixture)

0.75ul

RNA (template)

2ul

RTase (200U / ul) (reverse transcriptase)

0.5ul

DEPC H2O

to 20ul

[0072] B: Reverse transcription reaction conditions:

[0073] The reaction conditions are: 16°C for 30 minutes; 42°C for 30 minutes; 85°C for 10 minutes.

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Abstract

The invention discloses a group of oligonucleotides used for ankylosing spondylitis diagnosis and an application for detecting small RNA expression level thereof. The oligonucleotide comprises reverse transcription primer, probe, forward primer and reverse primer. The oligonucleotides comprise probes and primers for detecting the following small RNAs: hsa-miR-17-5P, hsa-miR-126-3P, hsa-miR-27-a and hsa-miR-29-a. The invention further provides a kit containing the oligonucleotides and a detection method for performing hybridization detection to the small RNA of ankylosing spondylitis patients;detection shows that the small RNA expression level of samples of the patients is higher than that of a normal person so that the oligonucleotides of the invention can be used as the diagnosis reagent of ankylosing spondylitis.

Description

technical field [0001] The present invention relates to the use of a group of micronucleic acids, in particular to the use of hsa-miR-17-5p, hsa-miR-126-3p, hsa-miR-27-a, hsa-miR-29-a as biomarkers , belongs to the technical field of biomedical materials. Background technique [0002] In recent years, a kind of non-coding single-stranded RNA small molecules (microRNA-miRNA) widely exist in cells from plants, nematodes to humans, and they can be incompletely complementary to specific regions of the 3' untranslated end of their target mRNAs. interact and inhibit protein synthesis. In 1993, Lee et al. cloned the lin-4 gene for the first time in C. elegans using the classical cloning method, and confirmed the existence of small molecule RNA by the method of point mutation. In 2000, Reinhart et al. discovered the existence of the let-7 gene in humans and Drosophila, and speculated that this small molecule may be a class of evolutionarily conserved regulatory molecules that play...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 古洁若张佩琢段春晓李志甲
Owner SUZHOU GENEPHARMA