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Method for measuring short chain RNA by amplifying length polymorphism of DNA fragment

Through the amplified DNA fragment length polymorphism quantitative determination method, the use of internal standards and dynamic small RNA standards without natural homologous sequences solves the sensitivity and standardization issues of miRNA quantitative determination and achieves highly accurate and comparable results. Suitable for clinical diagnosis and research of complex biological samples.

Active Publication Date: 2014-10-29
CHENGDU NUOEN BIOLOGICAL TECH
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Problems solved by technology

In addition, for the treatment and purification of samples, protein denaturants such as guanidine hydrochloride and phenol are generally used to obtain highly pure RNA. However, since the average size of miRNA is only 22nt, high-concentration ethanol is required for long-term precipitation to avoid loss. The contradiction of yield is more prominent, and the purification method of affinity resin column can generally guarantee the purity of RNA, but the integrity of purified RNA needs to be proved, that is, the loss and selective loss of miRNA

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  • Method for measuring short chain RNA by amplifying length polymorphism of DNA fragment
  • Method for measuring short chain RNA by amplifying length polymorphism of DNA fragment
  • Method for measuring short chain RNA by amplifying length polymorphism of DNA fragment

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Embodiment 1

[0060] Embodiment 1 miRFLP quantitative analysis method reaction process and measurement principle

[0061] This example takes omega primers as an example to specifically illustrate the reaction process and measurement principle of the miRFLP quantitative analysis method.

[0062] The miRFLP reaction consists of four steps: miRNA reverse transcription, cDNA tailing, PCR synchronous amplification, and fluorescent fragment length polymorphism analysis of PCR products, such as Figure 1A and Figure 1B shown. The first step of the reaction is to hybridize miRNA and omega primers, miRNA is paired with complementary probes, and then reverse transcriptase is used to synthesize cDNA using the unpaired miRNA 3' end as a template. After removing the RNA, the newly synthesized cDNA is hybridized with a 3' oligonucleotide primer containing a common PCR target and DNA polymerase is used to fill the single-stranded gap after DNA pairing starting from the 3' end of the cDNA. After this co...

Embodiment 2

[0064] Example 2 Determination of Fluorescent Intensity of PCR Fluorescently Labeled Product Equal Dilutions

[0065] The fluorescence intensity measured by the fluorescence quantitative analyzer is directly proportional to the amount of the fluorescent substance to be measured. This relationship is related to the fluorescence probe configured by the instrument, that is, different fluorescence probes have different fluorescence response curves. The fluorescence intensity measured by ABI's Prizma 310 DNA sequencer has a linear and quantitative relationship with the amount of fluorescent substance to be tested in the range of 5-7000 FU, and changes to a parabola when the fluorescence intensity value is greater than 7000FU. The response characteristics of fluorescence probes of different instruments are different, which can affect the regression relationship between the measured fluorescence intensity and the measured fluorescence amount. In this embodiment, the fluorescence resp...

Embodiment 3

[0067] Example 3 miRFLP quantitative analysis assay test of miR-92a and miR-92b

[0068] In the miRFLP analysis method, the miRNA to be tested and the dynamic miRNA standard are evenly mixed, and after miRNA reverse transcription, cDNA tail modification and fluorescent PCR synchronous amplification, DNA sequencer is used to analyze the length of DNA fragments and quantitative fluorescence. Specifically, first prepare 4 μl hybridization master mix: 2 μl 5xRT buffer, 1 μl 10 mM MgSO 4 , 1 μl dynamic miRNA standard mixture, the mixture includes the number of molecules is 3 × 10 6 The "Standard 1 RNA" (i.e. Figure 4A "std1", the same below), the number of molecules is ×10 5 The "Standard 2 RNA" (i.e. Figure 4A "std2", the same below), the number of molecules is 3×10 4 The "Standard 3 RNA" (i.e. Figure 4A "std3" in , the same below), the RNA sequence of the dynamic miRNA standard is shown in Table 2, and the sequence search of the Sangers miRbase20 database proves that ther...

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Abstract

The invention discloses a method for measuring a short chain RNA by amplifying length polymorphism of a DNA fragment and belongs to the l field of molecular biological techniques. The method comprises the following steps: first, by using at least two synthesized small RNAs without natural homologous sequences as compared with the short chain RNA to be measured as an interior label of measurement, mixing the synthesized small RNAs in different numbers of molecules to form a dynamic small RNA standard molecular gradient; and then, mixing with the short chain RNA to be measured in equal amount according to the dynamic small RNA standard, and carrying out RNA inverse transcription, cDNA tailing, PCR synchronous amplification and fluorescent quantitative analysis on length polymorphism fragment of DNA of the PCR product to measure a relative proportion of the fluorescence intensity of the DNA fragment amplified by short chain RNA to be measured in the dynamic small RNA standard fluorescence intensity gradient. The method can absolutely quantify the short chain RNA, particularly miRNA, has high specificity, high sensitivity, objective result and good repeatability, can realize accurate quantification in a copy number range of 100-10<6> and is also suitable for RNA crude extracts.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for quantitatively measuring short-chain RNA by amplified DNA fragment length polymorphism. Background technique [0002] microRNA (ie miRNA) is a kind of short single-stranded small molecule ribonucleic acid that regulates gene expression, consisting of about 22 nucleotides (nt). miRNA participates in various activities of cells, affects and participates in maintaining the physiological and differentiation state of cells, and changes in the expression of specific miRNAs can also reflect the physiological and pathological characteristics of organisms, indicating the possibility of using them as biomarkers for identifying various diseases. For example, changes in blood levels of miR-122, which is unique to liver cells, can reveal liver damage. miR-122 is unique to liver cells, and there are 130,000 in each human liver cell. It has specificity and sensitivity th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2535/138C12Q2545/114C12Q2525/207C12Q2537/165C12Q2545/101C12P19/34C12Q1/6855C12Q1/6869C12Q2600/16
Inventor 徐凯唐放张耀艺冯梓浩杨宇吴秀锦张菲菲
Owner CHENGDU NUOEN BIOLOGICAL TECH
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