FAM57B gene and expressed product thereof serving as target for diagnosis and treatment of bile duct cancer
A technology for expressing products and cholangiocarcinoma, which can be used in gene therapy, microbial determination/examination, biological testing, etc., and can solve the problems of low surgical resection rate and radical cure rate, and lack of diagnostic methods.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0053] Example 1 Screening of gene markers associated with cholangiocarcinoma
[0054] 1.1 Sample Collection
[0055] 10 samples of normal bile duct tissue and cholangiocarcinoma tissue were collected. The above samples are surgical resection specimens of patients with cholangiocarcinoma, and all the above samples were obtained with the consent of the organizational ethics committee.
[0056] 1.2 RNA sample preparation and quality analysis
[0057] 1.2.1 Preparation of RNA samples
[0058] Total RNA in advance using QIAGEN Tissue RNA Extraction Kit. Specific steps are as follows:
[0059] 1) In a clean area with less RNase interference, use a mortar containing an appropriate amount of liquid nitrogen to weigh about 20 mg of the isolated liver cancer tissue sample, and grind it to powder with a pestle;
[0060] 2) Transfer the sample to a RNase-free 2mL centrifuge tube;
[0061] 3) Add 300 μl Lysis solution, place in a homogenizer, and grind thoroughly for 1-5 minutes;
...
Embodiment 2
[0098] Example 2 QPCR sequencing to verify the differential expression of the FAM57B gene
[0099] 1. According to the detection results of high-throughput sequencing, the FAM57B gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 90 cases of cholangiocarcinoma tissue and 90 cases of normal bile duct tissue were selected.
[0100] 2. The RNA extraction steps are the same as in Example 1.
[0101] 3. Reverse transcription: use the reverse transcription kit of TAKARA company to operate. Specific steps are as follows:
[0102] (1) Take 2 μg of total RNA for reverse transcription, add 2 μl of Oligo(dT), and mix well. Immediately after 5 minutes in 70°C water bath, ice bath for 2-3 minutes.
[0103] (2) Construct a 25 μl reaction system, including 5 μl of 5× reverse transcription buffer, 5 μl of dNTP (2.5 mM), 40 U / μl of RNasin, 200 U / μl of M-MLV, and make up to the expected volume with nuclease-free water.
[0104] (3)...
Embodiment 3
[0124] Embodiment 3 inhibits FAM57B gene expression
[0125] 1. Cell culture: human cholangiocarcinoma cell line QBC939, with DMEM (high glucose) medium containing 10% calf serum at 37°C, 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. The medium was changed once every 2-3 days, and 0.25% trypsin was used for routine digestion and passage.
[0126] 2. siRNA design
[0127] siRNA sequence against FAM57B:
[0128] siRNA1-FAM57B:
[0129] The sense strand is 5'-UUGAACUUCUGGAAAUGUCUU-3' (SEQ ID NO.7);
[0130] The antisense strand is 5'-GACAUUUCCAGAAGUUCAAGC-3' (SEQ ID NO.8),
[0131] siRNA2-FAM57B:
[0132] The sense strand is 5'-UAUUAGCGGUCUGUAAAGCAC-3' (SEQ ID NO.9);
[0133] The antisense strand is 5'-GCUUUACAGACCGCUAAUAAA-3' (SEQ ID NO.10),
[0134] siRNA3-FAM57B:
[0135] The sense strand is 5'-AAUAAAUAAAAUAAAUAAGAU-3' (SEQ ID NO.11);
[0136] The antisense strand is 5'-CUUAUUUAUUUUAUUUUAUUUA-3' (SEQ ID NO.12)
[0137] Negative control siRNA ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More