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High-throughput sequencing method for absolute quantification of RNA molecules

An absolute quantitative, high-throughput technology, applied in the fields of molecular biology and biomedicine, can solve the problems of ligation efficiency preference that cannot be accurately quantified, RNA sequencing quantification that cannot be modified, etc., and achieve the effect of simple method

Active Publication Date: 2019-01-08
中磷科技深圳有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the inability to quantify modified RNA sequencing in the prior art, and the inability to accurately quantify due to ligation efficiency preference, the present invention provides a method for accurately quantifying intracellular RNA molecules. By designing a special DNA linker, the first time Realize the capture and absolute quantification of all RNA molecules in the sample, not only the absolute quantification of RNA samples from different sources, but also the quantification of RNA molecules containing modification sites

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  • High-throughput sequencing method for absolute quantification of RNA molecules
  • High-throughput sequencing method for absolute quantification of RNA molecules
  • High-throughput sequencing method for absolute quantification of RNA molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Linker1 efficiently connects tRNA samples

[0037] 1. RNA sample preparation

[0038] Monoclonal yeast was inoculated in 5 mL of YPD medium, cultured overnight at 30°C, and 1 mL of bacteria was collected, and RNA samples were extracted with the Purelinkessmall RNA isolation kit (Invitrogen, USA). The operation of the kit was performed according to the instructions.

[0039] 2. Dephosphorylation of RNA samples

[0040] Add 1 μL rSAP (NEB, USA) and 1 μL NEB cutsmart buffer to 50 ng RNA sample, add water to 10 μL, react at 37°C for 15 minutes, and stop the reaction at 65°C for 10 minutes.

[0041] 3. DNA Linker1 connection

[0042]Add 22 μL of the mixed solution (containing 100 pmol Linker1, 10 mmol ATP, 30 units of T4 RNA ligase 1 (NEB, USA), 2 μL buffer, 15 μL PEG8000) to 10 μL dephosphorylated RNA sample. After reacting overnight at 16°C, the reaction solution was purified with the ZymoOligo Clean & Concentrator kit, and the sample was eluted in 19 μL of wa...

Embodiment 2

[0043] Example 2 Linker 2 connects cDNA samples

[0044] 1. DNA sample preparation

[0045] Linker 2 samples were chemically synthesized (IDT, USA), and the sequence is as follows Figure 4 shown. cDNA samples were chemically synthesized DNA samples with a length of 60 nt (IDT, USA), and the sequences were random sequences to simulate cDNA samples from different sources.

[0046] 2. Linker 2 hairpin structure formation

[0047] Dissolve 10 μg Linker 2 in 10 μL water, denature at 95°C for 3 minutes, then cool down to 4°C at a rate of 0.1°C per second to form Figure 4 hairpin structure in .

[0048] 3. Ligation reaction

[0049] The 20 μL reaction system contains: 50 ng cDNA sample, add 1 μg (20 times) or 2.5 μg (50 times) Linker2, 2 μL T4 DNA ligase (NEB, USA), 2 μL buffer, 10 μL PEG8000, 22 ° C reaction After 12 hours, the reaction solution was purified with the ZymoOligo Clean & Concentrator kit, and the sample was eluted in 19 μL of water. bioanalyzer detects connect...

Embodiment 3

[0050] Example 3 Accurate Quantification of tRNA Molecular Changes in the Incubation Period of Mycobacterium tuberculosis

[0051] 1. RNA sample preparation

[0052] Mycobacterium tuberculosis BCG monoclonal cells were inoculated in 100 mL of 7H9 medium (Middlebrook), cultured on a shaker at 37°C for 36 hours, collected and suspended in 100 mL of PBS buffer. Take out 10mL suspension, centrifuge and collect thalli, extract RNA sample, this sample is the sample of incubation period 0th day (S0). The remaining suspension continued to be cultured at 37°C on rollers, and 10 mL of bacteria were taken out on the 4th, 10th, and 20th day of the culture, and the RNA samples were extracted, which were the samples on the 4th (S4), 10th (S10), and 20th (S20) days of the incubation period. Twenty days later, all the thallines were collected, and after 6 days of continuous cultivation with 7H9 medium, the thallines were collected, and the RNA samples were extracted, which was the 6th day (R...

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Abstract

The invention provides a high-throughput sequencing method for absolute quantification of RNA molecules. The method comprises the following steps: dephosphorylating a RNA sample; ligating the dephosphorylated RNA to DNA linkers 1 by T4 RNA ligase to capture 95% or more than 95% of RNA; then using demethylase to reduce RNA modification; removing excess linkers 1; reverse-transcribing to obtain cDNAwith the RNA as a template; degrading the RNA template; ligating the cDNA to DNA linkers 2 of a hairpin structure by the T4 DNA ligase so as to ligate 98% or more than 98% of the cDNA; removing excess linkers 2; and finally performing PCR primer amplification and sequencing. According to the invention, the defects of inaccurate quantification caused by the ligation efficiency preference in the existing RNA sequencing technology and the inability to capture RNA molecules containing modified sites are overcome by specially designing the DNA linkers, thereby realizing the absolute quantificationof all RNA molecules in RNA samples from different sources. The method provided by the invention is simple and can be used in the fields of clinical molecular diagnosis, molecular biological research, bacterial resistance, cancer occurrence and prevention and the like.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biomedicine, and relates to a method for absolute quantification of RNA molecules, in particular to a high-throughput sequencing method for determining the copy number of RNA molecules in a sample. Background technique [0002] With the development of molecular biotechnology, modern biology and biomedical research has entered the research stage of "omics", that is, to study the global metabolic network and operating mechanism in cells by capturing tens of millions of molecular dynamic information in cells , such as functional genomics, proteomics, metabolomics, etc. Among them, as the transmitter of genetic information, many other functions of RNA molecules in cells have gradually been revealed, such as regulating gene expression, biocatalysis, regulating protein translation efficiency, and cell drug resistance. [0003] With the development of next-generation sequencing technology (NGS, hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2535/122C12Q2525/191C12Q2521/501
Inventor 曹博
Owner 中磷科技深圳有限公司
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