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Extracting method for nasal cavity exfoliative cell RNA

A technology of exfoliated cells and extraction methods, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problem of unpublished and effective gene extraction methods for subtypes of chronic sinusitis, difficulty in obtaining real-time dynamic change information of diseases, and mucosal pathology in the non-healing period Biopsy and other problems, to achieve the effect of simple and fast extraction method, which is conducive to the protection of RNA and avoiding the risk of infection

Inactive Publication Date: 2018-11-30
张罗 +5
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Problems solved by technology

However, the existing method has the following problems: this method is an invasive examination, which increases the risk of infection for patients, and is not suitable for people with low immunity such as children and the elderly; nasal cavity bleeding often causes fear and anxiety in patients. Concern; in addition, it is difficult to obtain real-time dynamic change information of the disease through this method: pathological biopsy of the healing mucosa cannot be performed after the operation
However, in the prior art, the genes for effectively detecting the subtypes of chronic sinusitis with nasal polyps have not been screened, and further, there is no disclosure of the relevant methods for effectively providing the extraction methods for detecting and judging the subtypes of chronic sinusitis with nasal polyps. step
Therefore, in this field, there is no breakthrough in obtaining methods for detecting subtypes of chronic sinusitis with nasal polyps based on real-time fluorescent quantitative PCR, and there are major obstacles

Method used

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preparation example Construction

[0045] In an optional embodiment, the following steps are also included: the preparation method of the DNase reaction solution includes the following steps: get DNase buffer, recombinant DNase, and double distilled water to remove RNase to obtain the DNase reaction solution . Preferably, the preparation method of the DNase reaction solution comprises the following steps: take 5 μL of 10×DNase buffer solution, 4 μL of recombinant DNase, and 41 μL of RNase-removed double-distilled water and mix to obtain the DNase reaction solution.

[0046] In an optional embodiment, when the genome content is low or the initial amount of material is low during RNA extraction, the method for extracting RNA from exfoliated cells in the nasal cavity further includes the following steps: in the step 1, the The exfoliated cells from the nasal cavity were dissolved in the cell lysate and added to the genomic DNA adsorption column, and then centrifuged to obtain the supernatant.

[0047] In an optio...

Embodiment 1

[0065] Sample collection and processing:

[0066] After rinsing the nasal cavity with normal saline, a patient pressed a brush (manufactured by Copan) on the surface of the nasal polyp for 30 seconds under the nasal endoscope, rotated it 3 to 4 times, brushed the surface of the polyp, placed the brush in the cell lysate, Store at 4°C for short-term (less than 24 hours), or transfer to -20°C for long-term storage.

[0067] A method for extracting RNA in nasal cavity exfoliated cells, comprising the following steps:

[0068] Step 1: Dissolve the nasal exfoliated cells in 100 μL of cell lysate, add an equal volume of 70% ethanol, and use a pipette to mix the solution evenly; immediately add the mixture to the RNA purification column, 12000 rpm , centrifuged for 1min, removed the filtrate, and placed the RNA purification column in a 2mL collection tube;

[0069] Step 2: Add 300 μL of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for...

Embodiment 2

[0073] Sample collection and processing:

[0074] After rinsing the nasal cavity with normal saline, a patient pressed a brush (manufactured by Copan) on the surface of the nasal polyp for 30 seconds under the nasal endoscope, rotated it 3 to 4 times, brushed the surface of the polyp, placed the brush in the cell lysate, Store at 4°C for short-term (less than 24 hours), or transfer to -20°C for long-term storage.

[0075] A method for extracting nasal cavity exfoliated cell RNA, comprising the following steps:

[0076] Step 1: Take the genomic DNA adsorption column and place it in a 2mL collection tube, dissolve the nasal exfoliated cells in 300 μL of cell lysate, add them to the genomic DNA adsorption column, centrifuge at 12,000 rpm for 60 seconds, and take the filtrate. Add an equal volume of 70% ethanol to the filtrate, mix well and add to the RNA purification column, centrifuge at 12000 rpm for 1 min, remove the filtrate, and place the RNA purification column in a 2mL co...

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Abstract

The invention discloses an extracting method for nasal cavity exfoliative cell RNA. The extracting method comprises the following steps: putting nasal polyp exfoliative cells into a cell lysis solution to perform RNA extraction; brushing or pasting the nasal polyp exfoliative cells obtained on the surface of the nasal polyp, putting the obtained nasal polyp exfoliative cells into the cell lysis solution, storing for later use at a temperature not lower than 4 DEG C, and adopting an adsorption column process to extract the nasal cavity exfoliative cells RNA. A novel detecting method is constructed by a method for obtaining RNA from nasal cavity exfoliative cells, so that biopsy is avoided, and therefore, sampling can be performed at different follow-up stages, and patient disease course trace is performed. A noninvasive method is provided for obtaining nasal cavity epithelial cells, so that the infection risk of the patient is avoided.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a method for extracting RNA from nasal exfoliated cells. Background technique [0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammation of the sinus mucosa. Physical examination shows the formation of polyps in the nasal cavity or middle nasal passage. Common symptoms of CRSwNP are nasal congestion, runny nose or backflow, hyposmia, facial fullness or pressure, and last for more than 12 weeks. The prevalence rate is about 0.5-4%. CRSwNP is often accompanied by asthma and allergic rhinitis. It has been reported that 7% of asthmatic patients suffer from CRSwNP, while 26-48% of CRSwNP suffer from asthma. The pathogenesis of CRSwNP is still uncertain. The destruction of mucosal epithelial cells, the imbalance of host immune system and the invasion of pathogenic microorganisms may be the main causes of its pathogenesis. The main treatment modalities fo...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 张罗王成硕闫冰杨军
Owner 张罗
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