RNC binding agent and method for extracting RNC-RNA (ribosome nascent-chain complex and ribonucleic acid) from blood

A fixative and blood technology, applied in the field of life sciences, can solve problems such as difficulty in obtaining RNC, and achieve the effect of inhibiting activity and anti-oxidation

Active Publication Date: 2016-10-26
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of blood, which contains a large amount of endogenous and exogenous RNase and DNase, and RNC itself is very fragile, it is more difficult to obtain RNC in blood cells

Method used

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  • RNC binding agent and method for extracting RNC-RNA (ribosome nascent-chain complex and ribonucleic acid) from blood
  • RNC binding agent and method for extracting RNC-RNA (ribosome nascent-chain complex and ribonucleic acid) from blood
  • RNC binding agent and method for extracting RNC-RNA (ribosome nascent-chain complex and ribonucleic acid) from blood

Examples

Experimental program
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preparation example Construction

[0120] In the preparation method of the present invention, some of the reagents need to be formulated into a storage solution (mother solution) first, and then mixed to prepare a mixed solution for a specific purpose.

[0121] The water used is prepared with ultrapure water, and the prepared containers and suction tools are required to be RNase-free.

[0122] Ultrapure water: deionized water obtained by filtering with Millipore Synergy ultrapure water system, required level: 0.22μM filter membrane pore size, microorganisms <1cfu / ml: and sterilized by autoclaving at 121°C for 60min.

[0123] Use 1M HCl or 1M NaOH to adjust the pH of the solution or specific function mixture.

[0124] 1) RNC Blood Binding Solution components:

[0125] 150μg / ml cycloheximide, 30mM dithiothreitol (DTT),

[0126] 20 μM 1,2,3,4,6-O-pentagalloyl glucose (PGG), 30 mM sodium citrate (sodium citrate), 30 mM ethylenediaminetetraacetic acid (ED TA-2Na, pH 8.0), 15 mM ethylene glycol Bis(2-aminoethyl et...

Embodiment 1

[0136] The materials used in this example come from the General Hospital of Guangzhou Military Region, which are fresh whole blood from 7 breast cancer patients without chemotherapy and 2 normal people. Two tubes of blood were drawn for each sample, and the volume of each tube was 1 mL. One was used to extract RNCs and the other was used for biological replicates. The breast cancer group and the normal group are set. In this embodiment, the RNC-mRNAs of the two groups can be sequenced separately in order to study the differences in the expression of mRNA (RNC-mRNAs) translated by blood cells between breast cancer patients and normal people, and to judge the differences between different groups. Differences in protein types between proteins can provide a reference for mining drug targets.

[0137] Experimental process concrete steps of the present invention are as follows:

[0138] 1. Sample pretreatment

[0139] 1.1 Whole blood sample preparation

[0140] Two aliquots of fr...

Embodiment 2

[0182] The whole blood RNC-RNA of mammalian rats was extracted using the same set of reagents and protocols to compare the extraction effect of human whole blood.

[0183] The materials used in this comparative example were 10 fresh tail vein blood samples of healthy rats provided by the Experimental Animal Center of Sun Yat-sen University, with 2 samples for each sample, each with a volume of 1 mL. In subsequent experiments, one was used to capture RNC, extract RNC-RNA, and the other was used for biological repeats. Two copies of whole blood must be from the same sample source.

[0184] The concrete steps of present embodiment 2 are as follows:

[0185] 1 Sample pretreatment

[0186] 1.1 Whole blood sample preparation

[0187] Two parts of fresh whole blood were collected from the tail vein of rats using EDTA-containing anticoagulant tubes, each with a volume of 1 mL.

[0188] 1.2 RNC fixation and RNase inhibition: same as step 1.2 of Example 1

[0189] 2 RNC capture: sa...

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Abstract

The invention relates to an RNC binding agent and method for extracting RNC-RNA (ribosome nascent-chain complex and ribonucleic acid) from blood, wherein the RNC binding agent comprises the following components by content: 120-180 Mug/ml of cycloheximide, 25-35 mM of dithiothreitol, 0.45-0.65 M of ammonium chloride, 25-35 mM of sodium citrate, 25-35 mM of ethylene diamine tetraacetic acid, 10-20 mM of ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, 15-25 MuM of 1,2,3,4,6-O-pentagalloylglucose, 0.005-0.015% of glycerol, and the balance of water. The RNC binding agent (RNC blood binding solution) of the invention is a multifunctional reagent integrating translation extension inhibitor, RNA protectant and red blood cell lysate and may serve as an RNA protectant for use in the collection of conventional samples.

Description

technical field [0001] The invention belongs to the technical field of life sciences, in particular to an RNC fixative for extracting RNC-RNA from blood and an extraction method thereof. Background technique [0002] The "Central Dogma" states that genes are transcribed to produce mRNA, and mRNA is translated to produce protein, thereby completing the expression of genetic information. However, there is translation regulation during translation, and the correlation between mRNA abundance and protein abundance is generally low, so it is particularly important to study the translation status in cells. When the cell is translating, the ribosome size subunits are combined on the mRNA chain to move, and synthesize the protein polypeptide chain (nascent-chain) according to the mRNA codon information. This complex is called the ribosome nascent peptide chain complex RNC (ribosome nascent-chain complex), which is the basic unit of translation. All RNA combined on this complex is c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/125C12Q2527/127C12Q2565/113
Inventor 伍泳彰曾宏彬陈杰
Owner GUANGZHOU SAGENE BIOTECH
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