Unlock instant, AI-driven research and patent intelligence for your innovation.

Long non-coding RNA MYC-AS1 expression detection primer and kit

A technology of RNAMYC-AS1 and MYC-AS1, which is applied in the field of primers and kits for detecting the expression of long-chain non-coding RNAMYC-AS1

Pending Publication Date: 2020-07-24
YANGZHOU UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of DNA methylation inhibitors has also caused some confusion

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
  • Long non-coding RNA MYC-AS1 expression detection primer and kit
  • Long non-coding RNA MYC-AS1 expression detection primer and kit
  • Long non-coding RNA MYC-AS1 expression detection primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Determination of the full-length sequence of MYC-AS1.

[0028] (1) Identify the 5'and 3'end sequences of MYC-AS1;

[0029] Mainly provided by Takara RACE 5’ / 3’Kit kit to complete.

[0030] First, use Reagent extracts total RNA from human colon cancer cell line HCT116, and then removes the genome with RNase-freeDNase I. Under the action of SMARTScribeReverse Transcriptase (provided by the kit), 1μg of genome-removed RNA was reverse transcribed to synthesize 5'- or 3'-RACE products.

[0031] Then follow The operating instructions of RACE 5' / 3'Kit kit, use universal primer UPM and 5'-end or 3'-end gene-specific primer (gene-specific primer, GSP) for PCR amplification (RACE agarose gel) See electrophoresis figure 1 ), clone and sequence to obtain the 5'end and 3'end sequence of MYC-AS1. Among them, the nucleotide sequence of the 5'-end or 3'-end gene-specific primer used is as follows:

[0032] MYC-AS1-5-race:

[0033] 5'-CCACAGCAAACCTCCTCACAGCCCACT-3' (SEQ ID NO.6)

[...

Embodiment 2

[0044] Example 2 Construction of MYC-AS1 overexpression vector.

[0045] In Example 2, the MYC-AS1 full-length sequence obtained in Example 1 was used to construct a MYC-AS1 overexpression plasmid.

[0046] Specifically include the following steps:

[0047] (1) From the human colon cancer cell line HCT116, use Reagent extracts total RNA, and then removes the genome with RNase-free DNase I. It was reverse transcribed into cDNA product using PrimeScript RT reagent Kit.

[0048] (2) Using the cDNA product obtained in step (1) of Example 2 as a template, a high-fidelity enzyme was used to amplify the full-length sequence of MYC-AS1, wherein the primers MYC-AS1-HindIII-F and MYC-AS1-BamHI- The nucleotide sequence of R is as follows:

[0049] MYC-AS1-HindIII-F:

[0050] 5′-cccaagcttGCCTTTTCATTGTTTTCCA-3′ (SEQ ID NO.4)

[0051] MYC-AS1-BamHI-R:

[0052] 5′-cgcggatccCCTTTTTTAAGACGGAGTC-3′ (SEQ ID NO.5)

[0053] The reaction system includes: 100ng of the cDNA product obtained in step (1) of Examp...

Embodiment 3

[0056] Example 3 Analysis of the ability of MYC-AS1 to inhibit the expression of the proto-oncogene c-myc gene.

[0057] In this example 3, using the pcDNA3.1-MYC-AS1 overexpression plasmid obtained in example 2, the ability of MYC-AS1 to inhibit the expression of the proto-oncogene c-myc gene was evaluated by fluorescent quantitative PCR and Western-blot methods .

[0058] Specifically include the following steps:

[0059] (1) Inoculate the human colon cancer cell line HCT116 cells into a 6-well cell culture plate, and then transfect the pcDNA3.1-MYC-AS1 overexpression plasmid. At the same time, the pcDNA3.1-EGFP overexpression plasmid was transfected as a vector control.

[0060] (2) Collect cells 48 hours after transfection;

[0061] (3) Extract the total RNA of the cells and use the fluorescent quantitative PCR method to detect the expression level of the proto-oncogene c-myc in the cells.

[0062] (4) Extract the total cell protein, and analyze the expression level of proto-oncoge...

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

No PUM Login to View More

Abstract

The invention belongs to the field of molecular biology, and specifically relates to a long non-coding RNA MYC-AS1 expression detection primer and kit. The primer includes a chain specific RT primer and fluorescent quantitative PCR detection primers; the sequence of the chain specific RT primer is shown as SEQ ID NO. 8; and the sequences of the fluorescent quantitative PCR detection primers are shown as SEQ ID NO. 9 and SEQ ID NO. 10. The MYC-AS1 expression detection kit is also provided. Thus, methods and bases can be provided for the analysis of MYC-AS1 expression patterns; and the primer and kit can be used for detecting the expression situations of oncogenes in tumor samples.

Description

Technical field [0001] The invention relates to a primer and a kit for detecting the expression of long-chain non-coding RNA MYC-AS1, which belong to the research fields of epigenetics, oncology and new anti-tumor drugs. Background technique [0002] The occurrence of tumors is attributed to a variety of internal factors, such as genetic mutations, epigenetic mutations and metabolic abnormalities. It is generally believed that there are two main types of genes that are closely related to the occurrence of tumors, namely oncogenes and tumor suppressor genes. In normal cells, the expression of oncogenes and tumor suppressor genes maintain a certain balance. Both genetic and epigenetic mutations can cause abnormal expression of oncogenes and tumor suppressor genes, and promote normal cells to transform into cancer cells. [0003] Although genetic mutations are known to be related to the occurrence of cancer, more and more evidence shows that epigenetic abnormalities are also closely...

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
no application Login to View More
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 崔恒宓胡序明陈绪靖王丽萍
Owner YANGZHOU UNIV