Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for quickly and sensitively detecting micromolecule RNA

A sensitive detection, small molecule technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as bands cannot be displayed, background interference, and probes cannot be removed, so as to shorten the detection time and improve The effect of detection sensitivity and detection efficiency

Inactive Publication Date: 2014-12-24
SICHUAN UNIV
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot display bands, so it cannot distinguish small RNAs with similar sequences, and it is difficult to detect multiple small RNAs
Guo Yaohui et al. established a biotin-labeled-labeled method in "Biotin-labeled probe-liquid phase hybridization-non-denaturing PAGE method for detecting non-coding small RNA" (International Journal of Laboratory Medicine, Volume 33, Issue 6, March 2012). Liquid-phase hybridization-non-denaturing polyacrylamide gel electrophoresis is a method for detecting non-coding small RNAs. However, this method has a large background interference problem because it cannot remove redundant probes, and it is easy to produce false positive results that affect judgment, and it is difficult to achieve sensitivity. , accurate detection
In terms of actual detection effect, in order to obtain a better detection signal, the probe concentration required by this method is as high as 10 umol / L; and it takes a long time, from hybridization to detection of signal, excluding the time required for electrophoretic separation and membrane transfer. It takes 5 hours

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
  • Method for quickly and sensitively detecting micromolecule RNA
  • Method for quickly and sensitively detecting micromolecule RNA
  • Method for quickly and sensitively detecting micromolecule RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] In this example, the detection of Osa-miR156 in rice leaves, let-7-a in mouse hepatocytes, and chicken blood was taken as an example.

[0038]The flow chart of the whole detection method is as follows: figure 1 shown.

[0039] In the process of realizing the object of the present invention, the following steps are usually included:

[0040] 1. Material selection: fresh animal and plant tissues, blood, or fresh -70°C frozen samples.

[0041] 2. Reagent configuration:

[0042] 1) DEPC treated water RNase free water: deionized water is treated by Millpore water processor, add 1 / 1000 DEPC water for treatment, sterilize under high temperature and high pressure for 30 minutes, and cool to room temperature;

[0043] 2) TBE: Tris: 108g; Na 2 EDTA·2H 2 O: 7.44g; boric acid: 55g, add 800ml of deionized water, stir to dissolve, add deionized water to 1L, store at room temperature;

[0044] 3) 10% APS: 1g ammonium persulfate, dissolved in 10ml double distilled water;

[0...

Embodiment 2

[0083] Detection sensitivity test

[0084] This embodiment is based on the method of Example 1. During liquid phase hybridization, Osa-miR156 in rice leaves with different concentrations was used for liquid phase hybridization and solid phase detection, and the detection sensitivity test was carried out. The results are as follows: Figure 4 as shown, Figure 4The concentrations of Osa-miR156 in bands 1-15 are 1. 10 fmol; 2. 5 fmol; 3. 2.5 fmol; 4. 1 fmol; 5. 0.5 fmol; 6. 0.25 fmol; 7. 0.1 fmol; 8. 0.05 fmol; 9. 0.025 fmol; 10. 0.01 fmol; 11. 0.005 fmol; 12. 0.0025 fmol; 13. 1 amol;

[0085] from Figure 4 It can be seen that this method can detect at least 0.005 fmol of miRNA (5aM=35fg=0.035pg), that is, band 11 can be clearly identified, and the sensitivity of this method is 10 times higher than that of the previous small RNA detection method.

Embodiment 3

[0087] Small molecule RNA quantitative detection test

[0088] In this example, the quantification of miR156 in rice is taken as an example; the DNA form miD156 of miR156 with a series of concentration gradients (0.8-31.2 fmol) is hybridized with 0.1 pmol of biotin-labeled probe miD156*, and after color development, Chemi Doc The XRS detection system was used for relative quantitative analysis. The concentration of miD156 used for hybridization was used as the X axis, and the chromogenic concentration was used as the Y axis to establish a coordinate curve. The results showed a good linear relationship between the two. . Band 7 is the hybridization of 1ug rice miRNA with 0.1pmol miD156* probe, the result is in the regression equation,

[0089] The result is as Figure 5 as shown, Figure 5 In , the miR156 concentrations of bands 1-7 were 1: 0.8fmol; 2: 1.9fmol; 3: 3.9fmol; 4: 7.8fmol; 5: 15.6fmol; 6: 31.2fmol.

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 method for quickly and sensitively detecting a micromolecule RNA, and belongs to the field of molecular organisms. The method comprises the following steps: (1) extracting, and purifying a to-be-detected micromolecule RNA; (2) designing a probe, namely marking the probe by using biotin or digoxin; (3) liquid-phase hybridization, namely carrying out hybridization on the to-be-detected micromolecule RNA and the probe in a buffer solution, cutting off un-hybridized excessive single chain by using excision enzyme and then carrying out gel electrophoresis; (4) transferring a membrane, namely transferring the hybridized product on the gel after electrophoresis into a solid-phase support (such as nylon membrane, and a cellulose membrane); (5) incubating antibody, namely carrying out incubation hybridization on the solid-phase support containing the hybridized product obtained in the step (4) and a specific antibody; and (6) detecting a signal. According to the method, 0.005fmol of miRNA can be detected to the minimal extent, compared with the prior art, the sensitivity is improved by at least 10 times, the detection is rapid in detection speed, and high-through detection and quantitative detection can be achieved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for rapidly and sensitively detecting small molecule RNA. Background technique [0002] MicroRNAs (miRNAs) are a class of non-coding endogenous RNAs that have been found in multicellular species and even unicellular species. MicroRNAs can reflect gene expression from transcription to translation levels. Therefore, in different tissues and There are great differences in the expression levels of different developmental stages, and there are also great differences among different small RNAs. A series of research data have shown that small RNAs (miRNAs) can regulate gene expression at the post-transcriptional level through complementary base repair mode, and can also regulate gene expression at the DNA level by promoting DNA methylation. Therefore, small RNAs play important roles in almost all biological aspects including physiological, biochemical, developmental and pathol...

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): C12Q1/68
CPCC12Q1/6804C12Q2525/207C12Q2563/131
Inventor 王胜华毛强詹诚
Owner SICHUAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com