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A biological tissue RNA protection reagent and its preparation method and application

A protection reagent, a technology of sodium citrate, applied in the fields of biochemistry and molecular biology, can solve the problems that RNA cannot play a protective effect, and the DNA protection effect of isolated animal tissue is not ideal, so as to induce apoptosis of cancer cells, increase Transfection efficiency, enhanced phagocytosis effect

Inactive Publication Date: 2017-11-28
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the researchers also found that the solution only has a certain effect on protecting the RNA of animal tissues, but not the RNA of plant tissues, and the protection effect on the DNA of isolated animal tissues is not ideal.

Method used

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  • A biological tissue RNA protection reagent and its preparation method and application
  • A biological tissue RNA protection reagent and its preparation method and application
  • A biological tissue RNA protection reagent and its preparation method and application

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preparation example Construction

[0033] In the preparation method of the present invention, the preparation methods of the sodium citrate aqueous solution, EDTA aqueous solution, DTT aqueous solution, and PGG aqueous solution are as follows:

[0034] Prepare 1M sodium citrate aqueous solution: the molecular weight of sodium citrate is 294.1, weigh 44.1g sodium citrate in 100ml Milli-Q water and stir to dissolve, dilute to 150ml with Milli-Q water and store at 4℃.

[0035] Prepare 0.5M EDTA aqueous solution: EDTA·2Na·2H 2 O molecular weight is 372.24, weigh EDTA·2Na·2H 2 Dissolve 22.4g O2 in 75ml Milli-Q water with stirring, adjust the pH to 8.0 with 5N and 1N NaOH, dilute to 120ml with Milli-Q water, and store at 4°C.

[0036] Prepare 1M DTT aqueous solution: DTT molecular weight is 154.2, weigh 7.66g DTT in 40ml Milli-Q water, dilute to 50ml with Milli-Q water, and use it now.

[0037] Prepare 10mM PGG aqueous solution: PGG molecular weight is 940.68, weigh 94mg of PGG extracted from Radix Paeoniae Alba with a purity...

Embodiment 1

[0040] An RNA protection reagent, prepared according to the following method:

[0041] Add 2.2L Milli-Q water to a 5L sterilized glass bottle, add 1.74kg (NH 4 ) 2 SO 4 , Stir to completely dissolve, and then add the reagents prepared according to the above method in sequence: 62.1ml 1M sodium citrate aqueous solution, 50ml 0.5MEDTA aqueous solution, 50ml 1M DTT aqueous solution, 3.6ml 10mM PGG aqueous solution, and finally add 18ml glycerin and stir evenly , Use 1M H 2 SO 4 Adjust the pH to 5.2 at room temperature, dilute the volume to 3.6L with Milli-Q water, filter the prepared solution with a 1L (0.22um) filter, and store at 4°C.

[0042] The prepared RNA protection reagent A contains: 17 mM sodium citrate, 7 mM EDTA, 48% (W / V) ammonium sulfate, 14 mMDTT, 10 μM PGG, and 0.005% (V / V) glycerol.

Embodiment 2

[0044] An RNA protection reagent, prepared according to the following method:

[0045] Add 2.2L Milli-Q water to a 5L sterile glass bottle, add 1.44kg (NH 4 ) 2 SO 4 , Stir to dissolve completely, and then add the reagents prepared according to the above method in sequence: 54ml 1M sodium citrate aqueous solution, 72ml 0.5M EDTA aqueous solution, 72ml 1M DTT aqueous solution, 2.9ml 10mM PGG aqueous solution, finally add 3.6ml glycerin and stir Evenly, use 1M H 2 SO 4 Adjust the pH to 5.3 at room temperature, dilute to 3.6L with Milli-Q water, filter the prepared solution with a 1L (0.22um) filter, and store at 4°C.

[0046] The prepared RNA protection reagent B contains: 15 mM sodium citrate, 10 mM EDTA, 40% (W / V) ammonium sulfate, 20 mM DTT, 8 μM PGG and 0.001% (V / V) glycerol.

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Abstract

The invention provides an RNA (ribonucleic acid) protection reagent. The RNA (ribonucleic acid) protection reagent comprises the following components: 15-20mN of sodium citrate, 5-10mM of ethylene diamine tetraacetic acid, 40-55%W / V ammonium sulfate, 10-20mM of dithiothreitol, 8-15uM of 1,2,3,4,6-O-pentagalloylglucose and 0.001-0.010%V / V glycerinum. By utilizing the RNA protection reagent, in-vitro biological tissues can be stored for a period of time safely and conveniently at normal temperature without liquid nitrogen or carbon dioxide ice, and can be stored for a long time at low temperature; furthermore, the reagent is nontoxic and convenient in use, the used reagent can be directly poured into a water tank; compared with liquid nitrogen and carbon dioxide ice, the protection reagent provided by the invention is low in cost and is widely applicable. The invention further provides a preparation method and application of the RNA protection reagent.

Description

Technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and specifically relates to a chemical reagent for protecting RNA stability in isolated biological tissues. Background technique [0002] The information molecule RNA in the cell is a relatively fragile molecular substance, which is easily degraded by a variety of different mechanisms. Due to the widespread existence of RNase (Ribonuclease, RNase) and the stubborn nature of being difficult to inactivate, the extraction and purification of RNA and subsequent work have become very difficult. Operations involving RNA include two aspects: one is experimental operations (RNA extraction, RNA reverse transcription, RNA electrophoresis, etc.), and the other is RNA preservation. [0003] The main problem of RNA experiment operation is to prevent RNase contamination. RNase is everywhere. In any step of the experiment operation, any accidental negligence or improper operation may cause RNase co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/16C12N5/00A01N1/02A01N3/00
CPCA01N1/02A01N3/00C12N1/16C12N1/20C12N5/00C12N2500/72C12N2500/74
Inventor 吕昌龙翟景波高植鹏王大南姜雪峰
Owner 中国医科大学