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
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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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