Lysis solution and application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction

A lysate and tissue technology, applied in recombinant DNA technology, application, DNA preparation, etc., can solve problems such as low extraction efficiency and incompatibility of components, and achieve the effect of fast speed, stable properties, and eliminating the need for cleaning operations.

Active Publication Date: 2017-12-15
CHENGDU DAOSHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is: aiming at the incompatibility between the above-mentioned existing lysate for extracting RNA and the components of RNA later solution, which causes tissues or

Method used

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  • Lysis solution and application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction
  • Lysis solution and application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction
  • Lysis solution and application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039](1) Take eight 1.5ml EP tubes, add 4mg of rat liver tissue to each EP tube, and number each EP tube, among them, add 250ul lysate A to EP tubes 1, 3, 5, and 7 respectively, and Store at room temperature for 4h, 8h, 24h, and 48h respectively, add 250ul RNA later solution to EP tubes 2, 4, 6, and 8, and store at room temperature for 4h, 8h, 24h, and 48h respectively;

[0040] (2) Add 750ul lysate B to the EP tubes No. 1, 3, 5, and 7 above; discard the RNA later solution in the EP tubes No. 2, 4, 6, and 8 above, add 1×PBS to wash 3 times Discard the solution, and then add 1ml of TRIzol solution;

[0041] (3) The EP tubes No. 1-8 above were mixed upside down and subjected to ultrasonic cracking, the ultrasonic time was 5s, the frequency was 20kHz, and the power was 100W;

[0042] (4) Invert the ultrasonicated EP tube several times to mix the mixture evenly;

[0043] (5) Add 200 ul of chloroform to the EP tubes treated in step (4), and vigorously invert and mix for 30 s, ce...

Embodiment 2

[0051] (1) Take 12 1.5ml EP tubes and number each EP tube. Add 250ul lysate A and 750ul lysate B to EP tubes 1, 3, 5, 7, 9, and 11, respectively. , No. 6, No. 8, No. 10 and No. 12 EP tubes were added with 1ml TRIzol respectively;

[0052] (2) Put 4 mg of rat heart tissue into EP tubes 1 and 2, 3 mg of rat liver tissue into EP tubes 3 and 4, and 5 mg of rat liver tissue into EP tubes 5 and 6. Spleen tissue, put 4mg of rat lung tissue into EP tubes No. 7 and 8 respectively, put 5 mg of rat kidney tissue into EP tubes No. 9 and 10 respectively, put 3 mg of rat sciatic nerve tissue into EP tubes No. 11 and 12 respectively ;

[0053] (3) The EP tubes No. 1-12 above were mixed upside down and subjected to ultrasonic cracking, the ultrasonic time was 5s, the frequency was 20kHz, and the power was 125W;

[0054] (4) Invert the ultrasonicated EP tube several times to mix the mixture evenly;

[0055] (5) Add 200 ul of chloroform to the EP tubes treated in step (4), and vigorously inv...

Embodiment 3

[0063] (1) Take six 1.5ml EP tubes and add about 10 6 number each Panc-1 cell, and add 250ul lysate A and 750ul lysate B to EP tubes 1, 2, and 3 respectively, and add 1ml TRIzol to EP tubes 4, 5, and 6; Invert the EP tube several times to mix the mixture in it;

[0064] (2) Add 200ul of chloroform to the above-mentioned EP tubes, vigorously invert and mix for 10s, centrifuge for 1min under a centrifugal force of 10000g, and take the upper liquid;

[0065] (3) Pour the upper layer liquid obtained in step (2) into another batch of clean EP tubes with corresponding numbers, then add 500ul of isopropanol, slowly invert and mix for 50s, centrifuge at 10000g for 1min, discard To remove the liquid, to obtain a precipitate;

[0066] (4) Add 600ul of 75vt% ethanol solution to the above precipitate, slowly invert and mix for 30s, centrifuge for 1min under a centrifugal force of 10000g, and discard the liquid;

[0067] (5) Dry the EP tube in step (4) in the air for 5 minutes and add 4...

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Abstract

The invention discloses a lysis solution and an application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction. The lysis solution comprises a lysis solution A and a lysis solution B, wherein the lysis solution A comprises 2-4mol/L guanidinium isothiocyanate, 5-10mmol/L sodium chloride, 10-15mmol/L potassium chloride, the lysis solution B comprises 2-4mol/L guanidinium isothiocyanate, 1-5mmol/L spermidine, 30-40vt% of water saturated phenol, 0.4-0.5mol/L ammonium thiocyanate, 7-8vt% of glycerinum, 0.1-0.5mol/L sodium acetate and 0.2-0.5wt% of SDS (Sodium Dodecyl Sulfonate). When RNA is extracted by using the lysis solution A and the lysis solution B, tissue or cells do not need to be washed, and thus the extraction efficiency is remarkably improved; and due to synergistic effects of the components, the extracted RNA is high in purity and high in yield, and the purpose that animal RNA is extracted in laboratories rapidly with low cost and high quality in a large scale can be realized.

Description

technical field [0001] The invention belongs to the technical field of biological preparations, and in particular relates to a lysate and its application in preserving tissues or cells and extracting RNA. Background technique [0002] Extracting ribonucleic acid (RNA) is a technique often required in molecular biology experiments and clinical molecular diagnosis. The obtained RNA is widely used, including gene diagnosis, biochip analysis, gene expression analysis, etc. [0003] The storage method of tissue blocks directly affects the RNA extraction of animal tissue blocks as samples. The main storage methods are liquid nitrogen storage and RNA later solution storage (RNAlater TM Stabilization Solution). However, the chemical composition of the RNA later solution is not compatible with TRIzol, silica gel column and magnetic bead method. The RNA later solution contains ammonium isothiocyanate. When the tissue block is taken out from the RNA later solution for RNA extraction...

Claims

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Application Information

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IPC IPC(8): C12N15/10A01N1/02
CPCA01N1/021C12N15/1003C12N2310/10C12Q2527/125
Inventor 赵梓亦
Owner CHENGDU DAOSHENG BIOTECH CO LTD
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