Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A purification method and antisense technology, applied in the field of RNA antisense purification, can solve the problems of low product recovery efficiency, poor sensitivity and low efficiency, and achieve the effects of reducing non-specific adsorption, improving detection specificity and high sensitivity
Inactive Publication Date: 2017-05-31
GUANGZHOU BIOSENSE BIOSCI
View PDF2 Cites 4 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
[0005] However, the existing RAP technology has the disadvantages of low specificity and poor sensitivity due to problems such as magnetic beads will adsorb non-specific nucleic acids and proteins, the hybridization efficiency of probe sets and RNA is not high, and the recovery efficiency of nucleic acid and protein products is not high.
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0034] CTNNB1 is mRNA encoding catenin beta 1 protein, and its species is human. The RAP experiment was carried out for CTNNB1 mRNA, and the target gene was hsa-miR-214. The design of the probe and the method of labeling with biotin are well known to those skilled in the art, and will not be repeated here. Streptavidin coupled to magnetic beads (BeaverBeads TM Streptavidin, Beaver Biotechnology Co., Ltd.) and affinity binding with dethiobiotin; RNA was incubated with magnetic beads-RNA probe, and non-specifically bound RNA could be removed after washing; finally, the eluent (for strand Mycoavidin) for elution, RNA type can be verified after purification. Specific steps are as follows:
[0035] S1. Cell cross-linking, lysis and genomic DNA digestion to obtain RNA samples
[0036] S11, culturing and collecting cells
[0037] In this example, cCL-RAP cell culture or PAR-CL cell culture was used. A 15cm cell culture dish was used for culture, cCL-RAP cells were cultured in ...
Embodiment 2
[0066] SENP1 is mRNA encoding SUMO1 / sentrin specific peptidase 1 protein, and its species is human. The RAP experiment was carried out for SENP1mRNA, and the target gene was lincRNA-p21.
[0067] S1. Cross-linking and lysis of cells and digestion of genomic DNA to obtain RNA samples. Concrete method is identical with embodiment 1.
[0068] S2. Preparation of the probe set solution: the specific method for preparing the probe set solution in this example is the same as that in Example 1, except that the sequence of the probe set used in the experimental group is different.
[0069] In this example, the probe set of the experimental group contains 10 probes, and the probe set of the NC group contains 3 probes (the same as in Example 1). The specific sequence is shown in Table 3.
[0070] Table 1. Probe sequences for experimental groups
[0071]
[0072] S3, magnetic bead pretreatment: the same as in Example 1.
[0073] S4. Preparation of the probe set-magnetic bead compl...
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
Abstract
The invention relates to an RNA (Ribonucleic Acid) antisense purification method. The RNA antisense purification method comprises the following steps: S1, cross linking and pyrolysis of cells and digestion of genome DNA (Deoxyribonucleic Acid), thus obtaining an RNA sample; S2, preparation of a probe group solution; S3, pre-treatment of magnetic beads; S4, preparation of a probe group-magnetic bead compound; S5, antisense purification; S6, elution of RNA; S7, purification of the RNA. According to the RNA antisense purification method disclosed by the invention, the magnetic beads are sealed by using BSA (Bull Serum Albumin) and random oligonucleotide primer in advance, so that non-specific adsorption of the magnetic beads is reduced; the magnetic beads are in incubation with probes at first and then is in incubation with the RNA after residual probes are removed, non-specific combination of streptavidin with protein, nucleic acid and the like is sealed by excessive probes, the detection specificity is increased, and the sensitivity is high; multiple washing under a gradient temperature is carried out after hybridization, so that the magnetic beads are enabled to be fully combined with the probes and to be combined with a target RNA, and the hybridization efficiency is increased.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to an RNA antisense purification method. Background technique [0002] Long non-coding RNA (Long non-coding RNA, lncRNA) is a transcript of more than two hundred nucleotides, most of which are located in the nucleus. Although lncRNAs do not encode any protein, their expression is still specific in different tissues and developmental stages, which indicates that lncRNAs have important biological significance. With the help of sequencing technology, thousands of lncRNAs have been discovered, but the biological functions of these lncRNAs are still a mystery. [0003] In 2013, Professor Jesse M. Engreitz developed the RNA antisense purification (RNA antisense Purification, RAP) technology, which can identify the interaction of RNA with RNA and related DNA and protein at the post-transcriptional level. The principle is as follows: First, cells are fixed with ultraviolet cross-linking to ma...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more