MicroRNA standardization reference gene and application thereof

An internal reference gene and application technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, chemical library, etc.

Active Publication Date: 2013-07-17
MICROMEDMARK BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] No studies have systematically identified and evaluated the best reference genes for miRNA normalization. Therefore, there is an urgent need in the field to develo

Method used

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  • MicroRNA standardization reference gene and application thereof
  • MicroRNA standardization reference gene and application thereof
  • MicroRNA standardization reference gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0088] The preparation of the miRNA chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The miRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and placing it overnight for fixation.

[0089] Reagent test kit

[0090] The present invention also provides a kit comprising a reagent or a chip for determining an internal reference gene or an internal reference gene set, the internal reference gene or an internal reference gene set comprising 1, 2 or 3 microRNAs selected from the following group: let-7d, let-7g, and let-7i;

[0091] Wherein, described reagent is selected from following group:

[0092] (a...

Embodiment 1

[0115] Solexa sequencing and statistical analysis to screen the most stable reference gene

[0116] This example first generates and screens Solexa sequencing data sets to determine stable circulating miRNAs under various physiological and pathological conditions.

[0117] A total of 23 samples were analyzed and miRNAs meeting the following conditions were considered stable:

[0118] (1) expressed in all samples;

[0119] (2) High expression relative to the measured mean;

[0120] (3) The expression level was determined to be stable by measuring the standard deviation.

[0121] According to these criteria, 25 miRNAs were screened out as candidate reference genes, the results are shown in figure 2 .

[0122] Such as figure 2 As shown in A, the Solexa reads were converted to logarithmic values, and the genes were ranked by mean expression level and standard deviation; among all the miRNAs screened, 25 miRNAs had higher abundance in the data set (log 2 - Conversion reads ...

Embodiment 2

[0128] Screen for other candidate reference genes

[0129] This example further evaluates and analyzes the three candidate reference genes selected in the previous example and some commonly used reference genes. Commonly used reference genes include macromolecular RNA (GAPDH and β-actin), small nuclear RNA / nucleolus Small RNAs (snRNA / snoRNA) (U6, RNU44, and RNU48) and housekeeping miRNAs (miR-16, miR-191, miR-103, and miR-23a). Based on previous reports on their stable expression in tissues or cells, the inventors selected GAPDH and β-actin. U6, RNU44, RNU48, miR-16, miR-191, miR-103 and miR-23a were selected because they are often used as reference genes when measuring tissue / cell miRNAs. In addition, special attention was given to U6 and miR-16, as they have been used as reference genes for normalization of circulating miRNAs.

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Abstract

The invention relates to a microRNA standardization reference gene and an application thereof. Specifically, with Solexa sequencing, real-time fluorescence quantitative PCR detection, literature screening, statistical analysis methods, the invention carries out researches upon a large amount of samples collected from a healthy control group and different disease patients. As a result, microRNAs let-7d, let-7g, let-7i, or combinations thereof can serve as microRNA standardization housekeeping gene. Compared with reference genes (such as U6, RNU44, RNU48 and miR-16, and the like) most commonly used in current microRNA quantification, the microRNA standardization reference gene has high stability and accuracy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an internal reference gene for microRNA standardization and its use. Background technique [0002] MicroRNA (miRNA) is a class of non-coding RNA molecules with a length of about 22 nucleotides, which play an important role in gene regulatory networks. In body fluids such as serum, plasma, saliva, urine, and milk, miRNAs are highly stable outside the cells. miRNAs are associated with a variety of diseases, and the abnormal expression of miRNAs, especially circulating miRNAs, has been detected in a variety of disease conditions, including cancer, diabetes, heart failure, acute myocardial infarction, and tissue damage. In some pathological situations, especially in diseases like cancer, the expression profiles of miRNAs, especially circulating miRNAs, may change significantly with changes in physiological and pathological conditions. These studies demonstrate that miRNAs hold great pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/113C40B40/06
Inventor 张辰宇陈熹
Owner MICROMEDMARK BIOTECH CO LTD
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