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Method for directly detecting miRNA in absolute quantification mode

An absolute quantitative and direct technology, applied in the field of plasma miRNA quantitative detection, can solve the problem of inability to measure miRNA, and achieve the effects of improving sensitivity and repeatability, reducing method errors, and reducing the range of measurement variation.

Active Publication Date: 2016-02-17
CHENGDU NUOEN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes methods that use modified adapters or other agents to improve nucleic acid analysis techniques such as qPCR (quantitative polymerase chain reaction). These modifications help prevent unwanted signals from affecting certain parts of DNA being analyzed. Additionally, they enhance amplification efficiency during genetic testing procedures while also improving accuracy when measuring small amounts of mature microvesicles released into bodily fluids through brushing with blood samples collected on filter paper. Overall these improvements lead to increased precision and reliability in diagnostics tests performed today.

Problems solved by technology

Technologies described in this patents involve developing new ways to accurately determine specific nucleic acid sequences without interfering with existing techniques. These technical problem addressed include improving accuracy and reproduciblility during accurate measurements while minimizing variations among baseline values, reducing processing steps required, maintaining consistency standards across all test runs, simplifying preparations processes, providing confidence intervals for interpretation purposes, identiting unknown materials within the sample through mass spectrometry, and performing automated procedures based upon predilute genetic markers.

Method used

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  • Method for directly detecting miRNA in absolute quantification mode
  • Method for directly detecting miRNA in absolute quantification mode
  • Method for directly detecting miRNA in absolute quantification mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Effects of different concentrations of SDS, TritonX-100 and Tween-80 etc. on miRFLP assay

[0049] Many surfactants, such as SDS, Tween-20, TritonX-100, etc., can promote DNA synthesis at low concentrations, and are often used to increase the efficiency of RT or PCR reactions. High concentrations of surfactants inhibit the efficiency of enzyme reactions, but can also help dissolve cell membrane components and dissociate the tertiary structure of proteins. The inventors digested H1299 cells reaching 90% saturation in a 100 mm culture dish with trypsin, washed with PBS and divided them into 6 equal parts, added PBS, 0.5% SDS, 1% TritonX-100, 0.5% Tween-80, and carried out crack. After heating at 75°C for 5 minutes, quickly cool on ice, mix well and centrifuge at 10,000 g for 10 minutes, take the supernatant, and use miRFLP assay to measure miR-15a / b, miR-155, and miR-222. Dilute the sample so that the concentration of surfactant contained in each RT reaction ...

Embodiment 2

[0050] Example 2: Effect of 3' end-modified adapter oligonucleotide chains on miRFLP assays

[0051] Using the miRFLP assay to detect miR-150 / 146a / 222 / 146b, the adapter oligonucleotide chain used for cDNA elongation was used to add "AATTTAA" to the 3' end of the adapter oligonucleotide chain for three The results are listed in Table 1. Fluorescence intensity readings obtained with adapter oligonucleotides with modified 3' ends are generally lower than those obtained with adapter oligonucleotides with unmodified 3' ends. The modified adapter oligonucleotide chain has no primer function, which reduces the number of non-specific PCR templates and better maintains the linear relationship determined by the method. The detection of fluorescence intensity can be improved by increasing the number of PCR cycles or reducing the dilution factor to make up for the decrease in the detection limit caused by the decrease in the PCR template. Such as figure 1 As shown in B, mature miRNAs c...

Embodiment 3

[0054] Embodiment 3: The performance analysis of extracting-free miRFLP direct assay

[0055] The operation process of the comprehensively improved extraction-free miRFLP direct assay is as follows: the target miRNA to be tested is mixed evenly with the dynamic miRNA standard, after miRNA reverse transcription, cDNA tailing modification, biotinylation with agarose streptomycin coupling reagent The labeled omega products are enriched and purified, and then amplified simultaneously by fluorescent PCR. Finally, DNA sequencer is used to analyze the length of DNA fragments and quantitative fluorescence:

[0056] RNA denaturation solution: 5 μl 1% Tween-80, 200 μl 5xRT buffer, 100 μl ddH 2 O; dynamic molecular standard mixture; 2 μl RNA dynamic molecular standard (including: standard1RNA (dynamic molecular standard 1, and so on): 120,000; standard2RNA: 20,000; standard3RNA: 3340; standard4RNA: 600, the RNA sequence of the dynamic molecular standard See Table 12, its base sequences ...

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Abstract

The invention discloses a method for directly detecting miRNA in an absolute quantification mode, and belongs to the field of molecular biology. The method adopts an miRFLP measurement method and includes the steps that a sample to be detected and a dynamic miRNA standard material are mixed uniformly, miRNA reverse transcription, cDNA tailing, PCR synchronous amplification and fluorescence fragment length polymorphism analysis of a PCR amplification product are carried out, in the step of cDNA tailing, the tail end of an adaptive oligonucleotide chain 3' is modified, reaction background signals are reduced, and interference of homologous sequences at the tail end of miRNA 3' is avoided; a biotin-agarose streptomycin coupling reagent or streptomycin magnetic beads are adopted for enrichment of reverse transcription and PCR reactants, the loading quantity of the sample is increased, method errors are reduced, germ RNA is added as a protective reagent, and measurement errors are reduced. The sample of 0.4 microliter can be directly measured, and at the measurement level of 128 molecules, the changing range of measurement is reduced to 9.9%. The measurement sensitivity is remarkably improved, and the measurement error range is narrowed substantially.

Description

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Claims

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Application Information

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Owner CHENGDU NUOEN BIOLOGICAL TECH
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