A ribonucleic acid protecting agent, kit, application and preservation method
A ribonucleic acid and ribonuclease technology, applied in the field of molecular biology, can solve the problems of destroying the original biological activity of microRNA, extensive use of exRNA, difficulty in storage and transportation, loss of exRNA samples, etc.
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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 RNAlater 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 l...
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