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Method for Simultaneous Detection of Multiple RNAs

An annealing and systematic technology, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problem of no effective detection of miRNA and/or circRNA, and achieve the effect of lowering the detection limit and enhancing the sensitivity

Active Publication Date: 2022-04-15
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the prior art, there is no method for effectively detecting miRNA and / or circRNA, especially a method for simultaneously detecting multiple miRNA and / or circRNA

Method used

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  • Method for Simultaneous Detection of Multiple RNAs
  • Method for Simultaneous Detection of Multiple RNAs
  • Method for Simultaneous Detection of Multiple RNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] In order to illustrate that the present invention can detect miRNA and / or circRNA, this embodiment takes miR-21 as an example, and uses the detection method of the present invention to detect it, and the specific process is as follows:

[0067] miR-21 (sequence 5'-UAGCUUAUCUGUCUGAUGUUGA -3') was used as the detection target. Among them, NEase chooses Nt.BsmAI, and RNase inhibitor chooses Ribonuclease Inhibitor (sold by Promega), the cutting site is 5’-…GTCTCN↓N…-3’ in double-stranded DNA

[0068] 1. Design and synthesis of catch-21 and report-21 (catch-21 and report-21 are designed according to miR-21, the design principles and methods are as follows figure 2 shown, and artificially synthesized)

[0069] The sequence of catch-21 is:

[0070] 5'- CTGATGTT (GA GAC) TTTTT TCAACATCAG ACAGATAAGCTA-3'

[0071] The sequence of report-21 is:

[0072] 5'- / Cy5 / - GGA (GT CTC)AACATCACTG ACTCC - / BHQ / -3’

[0073] Wherein, the underlined part at both ends is the stem pa...

Embodiment 2

[0082] In order to prove the reliability and accuracy of the detection method of the present invention, this example uses the detection method of the present invention to detect miR-21 in the total RNA of cells. Among them, Nt.BsmAI is selected for NEase, and Nt.BsmAI is selected for RNase inhibitor Ribonuclease Inhibitor (sold by Promega).

[0083] The detection process is as follows:

[0084] 1. Extraction of cell total RNA

[0085] The cells used in the detection were cervical cancer cells Hela with high expression of miR-21 and normal liver cells L02 with low expression of miR-21.

[0086] The extraction method of cell total RNA is about 5×10 6 Add 1 mL of trizol reagent to the digested cells, incubate at room temperature for 5 minutes, then centrifuge at 12,000g and 4°C for 5 minutes. Add 200 μL of chloroform to the suspension, shake gently by hand, incubate at room temperature for 3 minutes, centrifuge at 12000 g, 4 degrees Celsius for 15 minutes, transfer the super...

Embodiment 3

[0092] In order to verify that the detection method of the present invention can detect different MiRNAs and / or circRNAs at the same time, this example uses the detection method of the present invention to detect multiple mixed samples containing different types of miRNAs. Among them, Nt.BsmAI is selected for NEase, and Nt.BsmAI is selected for RNase inhibitor Ribonuclease Inhibitor (sold by Promega). The detection process is as follows:

[0093] 1. The sequences used in this experiment are shown in Table 1. Among them, catch-21 and report-21 are catch probe and report probe designed and artificially synthesized according to miR-21, and the design method is as described above; catch-141 and report -141 designed and artificially synthesized catch probe and report probe based on miR-141, the design method is as above; catch-199a, report-199a designed and artificially synthesized catch probe and report probe based on miR-199a, the design method is as above ; catch-155, report-...

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Abstract

The invention provides a method for simultaneously detecting multiple RNAs, which is characterized in that it comprises the following steps: 1) designing a catch probe and a report probe; 2) annealing the catch probe and the report probe designed in step 1) respectively to form a hairpin structure; 3) Add the report probe after step 2) annealing, the catch probe after step 2) annealing, RNase inhibitor, NEase and RNA, incubate, then inactivate NEase, and detect the fluorescent signal in the system. The detection method of the present invention can detect samples containing multiple miRNAs, and simultaneously detect multiple miRNAs; it uses NEase to enhance fluorescence signals, thereby enhancing detection sensitivity and reducing the lower limit of detection.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for simultaneously detecting multiple RNAs, in particular to a method for simultaneously detecting multiple miRNAs and / or circRNAs. Background technique [0002] miRNA is an important non-coding single-stranded RNA containing about 18-24 nucleotides, which widely exists in animal and plant cells and plays an important role in many biological processes. More and more studies have shown that about 30% of human gene expression and translation are regulated by miRNAs. In addition, the expression levels of miRNAs are related to the occurrence and development of different tumors. Take miR-21 as an example, which is an oncogene and anti-apoptotic indicator expressed in breast cancer, prostate cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, colorectal cancer, glioma, cervical cancer and lung cancer . Therefore, miRNA has become one of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2525/207C12Q2525/301C12Q2563/107C12Q2527/127
Inventor 杜宇昊周翔张雅君
Owner WUHAN UNIV