Method for detecting SKA1 gene expression and purpose of siRNA thereof

A gene and target technology, applied in tumor diagnosis, the field of siRNA that inhibits the expression of SKA1 gene

Active Publication Date: 2013-08-21
上海尤里卡信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technical problem to be solved by the present invention is to address the lack of effective molecular diagnostic markers for predicting primary tumor drug resistance and molecular targets for treating primary tumor drug resistance in the clinical diagnosis and treatment of primary tumor drug resistance, and to provide An effective solution, that is, a group of siRNAs that can effectively inhibit the expression of the SKA1 gene, the target sequence of the siRNA and the reverse transcription PCR primers that can detect the expression of the SKA1 gene from tissues and cells and their use in the preparation or screening of anti- Application in Oncology Drugs

Method used

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  • Method for detecting SKA1 gene expression and purpose of siRNA thereof
  • Method for detecting SKA1 gene expression and purpose of siRNA thereof
  • Method for detecting SKA1 gene expression and purpose of siRNA thereof

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Experimental program
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Effect test

Embodiment 1

[0056] Example 1 siRNA design for SKA1

[0057] Through Invitrogen's online siRNA design software, four siRNA sequences with high evaluation on the website were selected. Through the NCBI website, the siRNA sequences were compared and analyzed (Blast analysis). These four siRNA sequences were found to have no significant homology to other genes except SKA1. The four siRNA sequences against SKA1 are listed in Table 1.

[0058] Table 1 SKA1 gene siRNA target sequence

[0059] Numbering

Embodiment 2

[0060] Example 2 Constructing a vector containing SKA1-siRNA

[0061] 2.1 Molecular synthesis

[0062] According to the sequences in the above table, the sense strand and antisense strand encoding siRNA were synthesized by Shanghai Sangon Biotech Co., Ltd. respectively. with ddH 2 O Dilute the synthesized sequence to a concentration of 100 μM for use.

[0063] The sequences of the four synthetic siRNA sense strands are:

[0064] 1. (SEQ ID NO.5):

[0065] 5'-CTAGCGGAGATGAGATCATTGTAATTCA AGAGATTACA ATGATCTCATCTCCTTTTTTGGAATTAAT-3';

[0066] 2. (SEQ ID NO.6):

[0067] 5'-CTAGCGAGGACTTACTCGTTATGTTATTCAAGAGATAACATAACGAGTAAGTCCTCTTTTTTGGAATTAAT-3';

[0068] 3. (SEQ ID NO.7):

[0069] 5'-CTAGCCCGCTTAACCTATAATCAAATTTCAAGAGAATTTGATTATAGGTTAAGCGGTTTTTTGGAATTAAT-3';

[0070] 4. (SEQ ID NO.8):

[0071] 5'-CTAGCGGGAGGACTTACTCGTTATGTTATTCAAGAGATAACATAACGAGTAAGTCCTCCCTTTTTTGGAATTAAT-3'.

[0072] The sequences of the synthesized 4 siRNA antisense strands are:

[0073] 5. (SEQ ID N...

Embodiment 3

[0125] Example 3 Virus packaging expressing SKA1-siRNA

[0126] 3.1 Plasmid intermediate extraction (intermediate extraction kit was purchased from Promega Biological Company, item number: A7640)

[0127] 1) Inoculate 50 μL of fresh bacterial liquid into 15-50 mL of LB medium (containing appropriate amount of antibiotics), and incubate with shaking at 37°C for 14-16 hours. Centrifuge at 5,000×g for 10 minutes at room temperature to collect the cells, and remove as much supernatant as possible.

[0128] 2) Add 2.5mL of BufferA1 to which RNase A has been added, and resuspend the cells.

[0129] 3) Add 2.5mL Buffer B1, invert gently 5-10 times to mix evenly, then let stand for 2-5 minutes until the solution becomes viscous and clear.

[0130] 4) Add 3.0mL Buffer C1, invert immediately 5 times, shake vigorously by hand 3-5 times to mix thoroughly, and white flocculent precipitate appears at this time.

[0131] 5) Transfer the centrifuge tube to a high-speed centrifuge and centr...

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Abstract

The invention discloses a siRNA for inhibiting SKA1 gene expression, and the length of the siRNA is 15-27 bp. The invention also discloses a recombinant vector containing siRNA, and the recombinant vector transfects the eukaryotic cell, and the cell with SKA1 gene silencing is obtained. The siRNA sequence can effectively degrade mRNA of SKA1 gene, thereby specifically inhibiting expression of SKA1 gene. The siRNA provided by the invention can be used for preparing medicaments for treating SKA1 gene related disease comprising tumour; the siRNA simultaneously can be used for researching effect of SKA1 gene in drug resistance of tumour, especially has a important meaning for clinic non-operation treatment of osteosarcoma and colorectum malignant tumor. The invention also discloses a reverse transcription PCR primer for effectively detecting SKA1 in tissue or cell, and applications of the primer in tumour diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a siRNA inhibiting the expression of SKA1 gene, the nucleotide sequence of the siRNA, a recombinant vector containing the siRNA coding DNA, and their application. The invention also relates to a reverse transcription primer capable of effectively detecting the expression of the SKA1 gene from tissues or cells, and the application of the primer in tumor diagnosis. Background technique [0002] RNA interference refers to the phenomenon of gene silencing induced by double-stranded RNA, which mainly inhibits gene expression by hindering the translation or transcription of specific genes. Studies have shown that small interfering RNA molecules (small interfering RNA, siRNA) with a length of 21-23nt are the direct cause of RNA interference (Tuschl T, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP- dependent cleavage of mRNA at 21 to 23 nucleotide inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113C12N15/63C12N7/01C12N5/10C12Q1/68C12Q1/02A61K48/00A61P35/00
Inventor 姚阳沈赞劳昕元余文熙
Owner 上海尤里卡信息科技有限公司
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