A non-freezing type RNA protection solution

A protective liquid, non-freezing technology, applied in the field of biology, can solve the problem of not being able to store samples in a low temperature environment, and achieve the effects of simple and convenient sample transportation, safe and reliable experimental operation, and simple operation.

Inactive Publication Date: 2017-03-15
宁波有成生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although freezing samples is a routine and effective method of sample preservation, in practice, it often happens that samples cannot be stored in a low-temperature environment in time; for example, clinical sample collection during surgery, field sample collection, sample transportation, etc. Ensure that the RNA molecules in the sample are not degraded at room temperature

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  • A non-freezing type RNA protection solution
  • A non-freezing type RNA protection solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Soak freshly dissected rat liver and kidney samples weighing about 200mg in 1mL RNA protection solution, place them in a sealed container at room temperature (25-35°C) for 14 days, take out the tissue, and remove the residual solution on the tissue with filter paper; Total RNA was extracted by Trizol method, and the integrity of RNA was identified by formaldehyde agarose denaturing gel electrophoresis.

[0030] The RNA extraction method is as follows: add 1 mL Trizol to 20 mg of tissue, fully homogenize with a disposable grinding rod, and after the sample is fully lysed, add 200 μL of chloroform, vibrate vigorously and mix well, then place at room temperature for 5 minutes to allow natural phase separation. Centrifuge at 12000r / min at 4°C for 10 minutes. The sample is divided into 3 layers, the yellow organic phase, the middle layer and the colorless aqueous phase. Transfer the aqueous phase to a new tube and add an equal volume of isopropyl alcohol that has been pre-coo...

Embodiment 2

[0043] Soak the freshly dissected rat liver weighing about 200 mg in 1 mL of RNA protection solution, place it sealed at room temperature (25-35° C.) for 7, 14, 28, and 42 days, and then extract total RNA using the Trizol method in Example 1. The dosage for each tissue extraction was 50 mg. After the extraction was completed, it was dissolved in 20 μL RNase-free water and stored at -80°C. After the last extraction was completed, the concentration and purity of the nucleic acid was detected by UV spectrophotometry. The test results are shown in Table 5. After the rat liver sample is immersed in the RNA protection solution of the present invention, when it is placed at room temperature for 14 days, the extracted RNA content has no significant difference compared with that of the fresh sample, and the RNA content decreases at 28 and 42 days.

[0044]Table 5: Purity and concentration of sample RNA at room temperature for different periods

[0045] Fresh 7 days 14 d...

Embodiment 3

[0048] Soak freshly collected samples of mulberry leaves, tomato leaves, and papaya leaves (about 200 mg each) in 1 mL of RNA protection solution, and place them sealed at room temperature (25-35°C) for 14 days. The purity of RNA was detected by spectrophotometry, and the detection results are shown in Table 6.

[0049] Grind the plant tissue thoroughly in liquid nitrogen, add 400 μL of the crushed tissue to a 2 mL centrifuge tube, and then add 1.2 mL of 65°C preheated extract (containing 50 mmol / L Tris-HCl pH 8.0; 10 mmol / L EDTA pH 8.0; 1mol / L NaCl: 2% SDS: 100mmol / L LiCl; 2% PVP40; 0.1% DEPC), shake and mix, add 600 μL of chloroform:isoamyl alcohol (24:1), shake and mix , centrifuge at 8000r / min at 4°C for 15 minutes, take the supernatant and add 400μL 8mol / L LiCl, mix well, precipitate at -20°C for 2h, centrifuge at 12000r / min at 4°C for 15 minutes, discard the supernatant, and wash with 500μL 0. 2M NaCl to dissolve the precipitate, add 500 μL of pre-cooled water-saturated...

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Abstract

The invention relates to a method for preserving the integrity of RNA (Ribonucleic Acid) in samples in the field of biology. The invention provides a non-freezing type RNA protection fluid which is safely operated and capable of preserving biological samples at normal temperature to enable the RNA of the biological samples to be protected from degrading. The biological samples capable of being preserved include human tissues, animal tissues, plant tissues, bacterium, fungi, culture cells and blood cells; after being dipped in the RNA protection fluid, the samples can be preserved for 14-42 days at room temperature (25-35 DEG C), and in the period, the RNA of the samples is kept in high integrity; the RNA with high quality can be extracted by using a conventional RNA extraction method. The non-freezing type RNA protection fluid is specifically suitable for the biological samples which cannot be preserved by freezing at low temperature immediately, such as the preservation of clinical samples, legal medical samples and field acquisition samples, the transportation of the samples, and the like.

Description

technical field [0001] The invention relates to a method for preserving the integrity of RNA in biological samples in the field of biology, in particular to a method for preserving biological samples at room temperature to prevent RNA from degrading. [0002] technical background [0003] Extraction and purification of intact, undegraded RNA molecules is a prerequisite for research and detection of RNA. Molecular biology experiments such as Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR, cDNA synthesis, and in vitro translation all require the use of high-quality, intact RNA molecules. Common biological samples used to extract RNA include human tissue, animal tissue, plant tissue, artificially cultured cells (animal cells, yeast cells, bacterial cells), blood cells, and the like. Due to the threat of degradation of RNA by the endogenous RNA decomposing enzyme RNase in the tissue and the exogenous RNase in the environment, f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/04C12N15/11A01N1/02C12Q1/68G01N21/64
Inventor 周细武邱英华牛欢欢
Owner 宁波有成生物医药科技有限公司
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