RNA protective agent, kit, application and preservation method

A ribonucleic acid and preservation method technology, applied in the field of molecular biology, can solve the problems of RNA fragmentation, extensive use of exRNA, difficulties in storage and transportation, loss of exRNA samples, etc.

Active Publication Date: 2016-11-02
上海晟燃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exRNA is often degraded by RNase after long-term storage, freeze-thawing and purification, which brings great difficulties to the widespread use, storage and transportation of exRNA
[0004] In the practical application of RNA, the preservation of RNA depends on pure water without RNase or an aqueous solution with chelating agents such as EDTA added to dissolve the RNA and store it in an ultra-low temperature freezer at -80°C for one year. For example, irreversible RNA fragmentation will be caused during freezing at room temperature to -80°C, and physical damage will destroy the original biological activity of microRNAs when returning to room temperature at -80°C.
[0005] There are currently several comm

Method used

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  • RNA protective agent, kit, application and preservation method
  • RNA protective agent, kit, application and preservation method
  • RNA protective agent, kit, application and preservation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The configuration of various ribonucleic acid protecting agents of embodiment 1

[0056] 1. Configuration of trehalose solutions with different concentrations

[0057] serial number Trehalose (g) Pure water (ml) Concentration (M) 1 4.62 50 0.27 2 5.13 50 0.3 3 6.85 50 0.4 4 8.56 50 0.5 5 17.11 50 1

[0058] Table 1

[0059] 2. Trehalose solution with ribonuclease inhibitor added

[0060] serial number RNase inhibitor (μl) Trehalose solution (0.5M, μl) volume concentration 1 0.1 2000 1:20000 2 0.2 2000 1:10000 3 0.4 2000 1:5000 4 1 2000 1:2000

[0061] Table 2

[0062] 3. Trehalose solution with ribonuclease inhibitors and EDTA

[0063]

[0064] table 3

[0065] 4. Trehalose solution with ribonuclease inhibitor and TE buffer

[0066]

[0067] Table 4

[0068] Note: *a. Tris-EDTA buffer is referred to as TE buffer (contains 10mM Tris, 1mM EDTA pH 8.0).

[0...

Embodiment 2

[0071] Example 2 Purification, Quantitative Determination and Integrity Analysis of ExRNA in Cultured Tumor Cells:

[0072] H332, SW1910 or Hela cells were cultured in 90mm culture dishes in RPMI medium containing 10% FBS in 5% CO 2 Cultivate to 90% saturation in a 37°C constant temperature incubator. After 48 hours, the cell culture fluid was collected, and 500 μl of Trizol reagent (LifeTech) was added to each 1 ml of the culture fluid, which was blown repeatedly with a pipette and allowed to stand at room temperature for 5 minutes. Subsequently, 200 μl of chloroform was added, vigorously shaken for 15 seconds, and then allowed to stand at room temperature for 10 minutes. Centrifuge at 12000g at 4°C for 15 minutes, draw the supernatant into a new EP tube, add an equal volume of isopropanol, blow and mix with a pipette, and store at -20°C overnight. The supernatant was removed after centrifugation at 12000g for 15 minutes at 4°C. Add 500 μl of 75% ethanol solution to shake ...

Embodiment 3

[0074] Example 3 Long-term preservation of exRNA by different commercial protective agents and trehalose solutions at -20°C

[0075] The exRNA purified from SW1910 cells by the method described in Example 1 was dissolved in RNA later solution of LifeTech, RNA stable solution of Biomatrica and 0.5M trehalose solution, and the concentration of exRNA was measured by ultraviolet spectrophotometer. The concentration of exRNA dissolved in RNA later is 13.7ng / μl, and the OD260 / 280 is 1.91; the concentration of exRNA dissolved in RNA stable is 17.9ng / μl, and the OD260 / 280 is 1.97; the exRNA dissolved in 0.5M trehalose solution Concentration is 21.2ng / μl, OD260 / 280 is 1.99 ( figure 1 and 2 ). Freeze-dry the exRNA stored in RNA stable and the exRNA stored in 0.5M trehalose into a solid state, and store the exRNA stored in RNA later in a liquid state at -20°C for 18 months. The pure water is dissolved to the volume before lyophilization, and the exRNA stored in RNA later returns to a ...

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Abstract

The invention provides an RNA protective agent, a kit containing the protective agent, application and a preservation method of RNA. The protective agent contains a trehalose solution with a concentration of 0.27M-1M. The protective agent provided by the invention can achieve the effects of long preservation time, temperature insensitivity and wide range of use temperature, an extracellular RNA sample treated by the protective agent has no influence to cDNA synthesis, PCR reaction and enzyme activity in sequencing and bank building. The protective agent provided by the invention can efficiently solve the RNA degradation, pollution and other problems encountered during long-term preservation or use of exRNA, and especially has important significance for preservation, repeated use and long-distance transportation of extracellular microRNA.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an RNA protection agent, a kit, an application and a preservation method. Background technique [0002] Ribonucleic acid (abbreviated as RNA, that is, Ribonucleic Acid), the genetic information carrier that exists in biological cells and some viruses and viroids, RNA is a long chain molecule formed by ribonucleotides condensed through phospholipid bonds, and can participate in The various life activities of cells are the key research objects in the fields of medicine, biology and pharmacy. [0003] With the in-depth study of RNA, it is found that cells with vital activities can secrete specific RNA from the cell to the circulatory system or specific tissue microenvironment, and this type of RNA is defined as extracellular RNA (exRNA). Due to the obvious difference between exRNA and common RNA in cells, exRNA can play various potential biological roles as a signal. At present, t...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王家亮王昉炜李辉辉刘月星
Owner 上海晟燃生物科技有限公司
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