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Efficient and stable bacterium total RNA (Ribonucleic Acid) extraction method

An extraction method, a bacterial technology, is applied in the field of microbial total RNA extraction, which can solve the problems of time-consuming, large differences, and poor extraction effects.

Inactive Publication Date: 2018-05-15
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, it must be pointed out that for different bacteria, the results presented by the same method often vary greatly
There are many published methods for extracting bacterial RNA, but most methods not only use some toxic reagents, but also are time-consuming, complicated, expensive, and the extraction effect is not good

Method used

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  • Efficient and stable bacterium total RNA (Ribonucleic Acid) extraction method
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  • Efficient and stable bacterium total RNA (Ribonucleic Acid) extraction method

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Embodiment 1

[0031] Embodiment 1: the extraction of Listeria monocytogenes RNA, described method specifically comprises the following steps:

[0032] The first step is as follows: prepare sterile TSB liquid medium, add Listeria monocytogenes with 1% inoculum, take out the bacterial liquid after shaking the bacteria at 37°C for 8 hours; place at 4°C for 10min; centrifuge at 5000×g After 10 minutes, the supernatant was discarded.

[0033] The second step is as follows: 200μL 15mg / mL lysozyme, 1mL blue tip to absorb appropriate glass bead powder into the extraction tube, vortex to mix, Tissue Cell-Destroyer DS1000 treatment, and its parameters are: Execute the project A, speed 5000RPM; time 30s; interval 10s; times 15.

[0034]The step three is specifically as follows: the kit used for subsequent processing is Bacterial RNA Kit R6950 (OMEGA), and 350 μL Buffer BRK / β-Me was added to the crushed solution, and vigorously vortexed for 5 minutes. Centrifuge at 13,000×g for 5 min; transfer 400 μL...

Embodiment 2

[0035] Embodiment 2: the extraction of Staphylococcus aureus RNA, described method specifically comprises the following steps:

[0036] The first step is as follows: prepare sterile TSB liquid medium, add Staphylococcus aureus to it with 3% inoculum, shake the bacteria at 37°C for 2h20min, then take out the bacterial liquid; place at 4°C for 10min; centrifuge at 5000×g for 10min, Aspirate and discard supernatant.

[0037] The second step is as follows: 200μL 15mg / mL lysozyme, 1mL blue shampoo, absorb appropriate glass bead powder into the extraction tube, vortex to mix, Tissue Cell-Destroyer DS1000 treatment, and its parameters are: Execute the project A, speed 5000RPM; time 30s; interval 10s; times 15.

[0038] The step three is specifically as follows: the kit used for subsequent processing is Bacterial RNA Kit R6950 (OMEGA), and 350 μL Buffer BRK / β-Me was added to the crushed solution, and vigorously vortexed for 5 minutes. Centrifuge at 13,000×g for 5 min; transfer 400 μ...

Embodiment 3

[0039] Embodiment 3: the extraction of Klebsiella pneumoniae RNA in the blood sample, described method specifically comprises the following steps:

[0040] The step 1 is specifically as follows: draw 2 mL of blood sample into a 15 mL centrifuge tube with a 5 mL disposable syringe, add ACKLysing Buffer, ice-bath for 30 min, and mix by inverting up and down every 5 min. 12000rpm / min, centrifuge for 10min, discard the supernatant. Resuspend the pellet in TE buffer and vortex to mix. Centrifuge at 5000×g for 10 minutes, discard the supernatant.

[0041] The second step is as follows: 300μL 15mg / mL lysozyme, 1mL blue tip to absorb appropriate glass bead powder into the extraction tube, vortex to mix, Tissue Cell-Destroyer DS1000 treatment, and its parameters are: Execute the project A, speed 5000RPM; time 30s; interval 10s; times 15.

[0042] The step three is specifically as follows: the kit used for subsequent processing is Bacterial RNA Kit R6950 (OMEGA), and 350 μL Buffer BR...

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Abstract

RNA (Ribonucleic Acid) is an important genetic substance in a living organism. The RNA extraction is the basis of molecular biology study; high-quality and high-completeness RNA is needed for downstream molecular biological experiments such as cDNA library construction, in vitro inverse transcription, real-time fluorescent quantitative PCR and Nothem hybridization assay. The project has the main content of providing an efficient bacterial cell wall breaking method; the cell breaking is more complete; the RNA dissolution is more facilitated; the high-quality total RNA capable of being directlyused for the downstream molecular biological experiments is obtained. The method has wide applicability. The bacterium belongs to a gram-positive bacterium. Compared with the other methods, the methodhas the advantage that the extracted RNA concentration is improved by 4 to 5 times. The work innovation is shown in aspects that during the bacterium treatment, Tissue Cell-Destroyer DS1000, lysozymeand glass beads are combined, so that the bacterial cell wall can achieve a good crushing effect; through the use of the Tissue Cell-Destroyer DS1000, the RNA extraction effect is greatly improved.

Description

technical field [0001] The invention designs an efficient and stable method for extracting microbial total RNA, which belongs to the technical field of nucleic acid extraction. Background technique [0002] It is an important genetic material in organisms. The extraction of RNA is the basis of molecular biology research. Good quality is required for downstream molecular biology experiments such as cDNA library construction, in vitro reverse transcription, real-time fluorescent quantitative PCR, and Nothem hybridization analysis. , RNA with high integrity. The extraction of high-purity and complete total RNA is the basis for gene transcription level manipulation, the study of bacterial virulence gene expression and its pathogenicity and environmental adaptability, as well as bacterial drug resistance genes and genes related to biofilm formation. It is the basis for ensuring the PCR reaction A prerequisite for normal operation. [0003] The essence of the extraction is to re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 万逸刘红黄帅
Owner HAINAN UNIVERSITY
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