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Nucleic acid composition for extracting or detecting small molecule RNA (Ribonucleic Acid) in sample as well as kit and method thereof

A technology of nucleic acid composition and small and medium molecules, which is applied in the field of nucleic acid detection, can solve the problems of large sample consumption, inability to effectively distinguish small molecule RNA, and inapplicability to large-scale screening experiments, and achieve high-throughput and accurate detection.

Pending Publication Date: 2022-03-29
BERRY ONCOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Northern blotting is a common method for detecting RNA based on hybridization. Its disadvantages are: it is not suitable for large-scale screening experiments, it cannot effectively distinguish small RNA molecules with subtle sequence differences, and the detection process is time-consuming and consumes a large sample size. Within 3 working days, 5-10 μg of total RNA is required to be successfully detected, and it is not suitable for the detection of traces of small RNAs from plasma and other sources; microarray analysis uses high-density fluorescent-labeled probes to hybridize with RNA samples, through fluorescence Scan to obtain expression map
[0010] In addition, due to the complexity of the composition of biological samples, most of the existing RNA detection methods need to extract the total RNA first, which greatly increases the detection cycle and labor costs, and there are also RNA degradation and target gene loss during the RNA purification process. risk

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  • Nucleic acid composition for extracting or detecting small molecule RNA (Ribonucleic Acid) in sample as well as kit and method thereof
  • Nucleic acid composition for extracting or detecting small molecule RNA (Ribonucleic Acid) in sample as well as kit and method thereof
  • Nucleic acid composition for extracting or detecting small molecule RNA (Ribonucleic Acid) in sample as well as kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] A kit for extracting or detecting small molecule RNA in a sample, comprising a nucleic acid composition, the nucleic acid composition includes silicon-based magnetic beads (solid phase carrier), capture probes and blocking probes.

[0084](1) Capture probes and magnetic beads

[0085] A single-stranded oligonucleotide (SEQ ID No. 1:5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTT-3') is immobilized on the magnetic beads.

[0086] The capture probe is a single-stranded DNA sequence, specifically including a first sequence and a second sequence connected to each other. The first sequence includes a reverse complementary sequence to the single-stranded oligonucleotide, and the second sequence can reverse complementary to the target gene or part thereof. It should be noted that the first sequence may also include a connecting sequence, and when the connecting sequence is included, the sequence reversely complementary to the single-stranded oligonucleotide is connected to the second sequenc...

Embodiment 2

[0109] A method for extracting target small molecule RNA in a sample and constructing a sequencing library, specifically comprising the following steps.

[0110] (1) Sample extraction

[0111] Taking plasma samples as an example, to extract small molecule RNA from biological samples: take -20°C or -80°C cryopreserved or freshly separated plasma, and add the extraction solution and proteinase K to a low-adsorption centrifuge tube in turn. For the specific reagent volume, please refer to Table 3, after mixing well, incubate at 65°C for 20min.

[0112] Table 3 volume of extract and proteinase K

[0113]

[0114] (2) Denaturation of the capture probe pool: The target gene capture probe pool is a mixture of multiple probes, and the probes may form secondary structures by themselves or with each other, which must be denatured before use to open the secondary structure. Take an appropriate amount of the probe pool, denature at 95°C for 2 minutes, and immediately place it on ice ...

Embodiment 3

[0121] Taking plasma samples as an example, this example provides a method for co-extracting Poly A-structured RNA (mRNA) and small molecule RNA and constructing a sequencing library. Using the kit provided in Example 1, first pass Oligo dT magnetic beads The dT sequence specifically captures the polyA structural RNA (mainly mRNA and lncRNA) in the lysed plasma sample, separates the polyA structural RNA through the first magnetic suction, and then adds the capture probe and Oligo dT magnetic beads described in this patent , referring to the method in Example 2, the small molecule RNA is captured through the second magnetic attraction, and the specific steps are as follows.

[0122] (1) Take -20°C or -80°C cryopreserved or freshly separated plasma, add the extract and proteinase K to the low-adsorption centrifuge tube in turn, the specific reagent volume can refer to Table 3, mix well, and incubate at 65°C for 20 minutes .

[0123] (2) Add the diluent in turn. The system can r...

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Abstract

The invention discloses a nucleic acid composition for extracting or detecting small molecule RNA in a sample and a kit and a method thereof, and relates to the technical field of nucleic acid detection.The nucleic acid composition comprises a solid-phase carrier, a capture probe and a closed probe, and single-stranded oligonucleotide is fixed to the solid-phase carrier; the capture probe comprises a sequence which is reversely complementary with the single-stranded oligonucleotide and a sequence which is reversely complementary with a target gene; the closure probe is capable of reverse complementation with the capture probe or portions thereof. The nucleic acid composition can capture and separate trace target small molecule RNA in a sample and construct a sequencing library under the condition that total RNA does not need to be extracted, a product of the nucleic acid composition can be directly applied to high-throughput sequencing detection, the nucleic acid composition has the advantages of rapidness, sensitivity and high efficiency, and accurate detection of various small molecule RNA can be achieved in a high-throughput mode.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid composition for extracting or detecting small molecule RNA in a sample, a kit and a method thereof. Background technique [0002] A large number of studies have shown that circulating free nucleic acid (cfNA) in the blood of healthy people and cancer patients has clinical application value as a new type of non-invasive diagnostic biomarker. [0003] Small RNA (Small RNA) is a class of highly conserved RNA molecules with a length of less than 50 nt. Acting RNA (piRNA), repeat-associated siRNA (rasiRNA), etc. They control gene expression at the transcriptional level, post-transcriptional level, and epigenetic level, and are widely involved in the regulation of organisms through a variety of pathways, including mRNA degradation, translational repression, heterochromatin formation, and DNA removal. growth and disease occurrence. [0004] Micro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 王源舒林明芳王岩杨浩王寅吴琳
Owner BERRY ONCOLOGY CO LTD
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