Marker for diagnosing and treating rectal adenocarcinoma
A rectal adenocarcinoma and drug technology, applied in gene therapy, biological testing, drug combination and other directions, can solve the problems of difficult to find, lost the opportunity of surgery, and hidden clinical symptoms of rectal adenocarcinoma.
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Embodiment 1
[0061] Example 1 Screening for gene markers associated with rectal adenocarcinoma
[0062] 1. Collection of samples
[0063]8 cases of rectal adenocarcinoma non-metastatic tissue and rectal adenocarcinoma metastatic tissue samples were collected, and all the above samples were obtained with the consent of the organizational ethics committee. All surgical resection specimens were cut into small pieces with sterilized ophthalmic scissors, put into 1.5ml sterile EP tubes soaked in 0.1% DEPC water overnight and sterilized, and the remaining tissues were stored in cryopreservation tubes and quickly put into Store in a liquid nitrogen tank at -80°C.
[0064] 2. Preparation of RNA samples
[0065] 1) Sample pretreatment: Take the sample from the -80°C liquid nitrogen tank and put it into a homogenizer soaked in 0.1% DEPC water overnight and sterilized, and grind it into powder; during the grinding process, the sample must always be in the In a liquid nitrogen environment, the grin...
Embodiment 2
[0085] Example 2 QPCR sequencing to verify the differential expression of SLC26A9 gene
[0086] 1. According to the detection results of high-throughput sequencing, the SLC26A9 gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 70 cases of non-metastatic rectal adenocarcinoma tissue and 70 cases of metastatic rectal adenocarcinoma tissue were selected.
[0087] 2. The RNA extraction steps are the same as in Example 1.
[0088] 3. Reverse transcription:
[0089] 1) Reaction system:
[0090] Reagent
volume
MgCl 2
2μl
10×RT Buffer
1μl
RNase-free water
3.75μl
dNTP mix
1μl
RNase inhibitor
0.25μl
0.5μl
Random 9mers
0.5μl
experimental sample RNA
1μl
[0091] 2) Reverse transcription reaction conditions
[0092] According to the reverse transcription reaction conditions in RNAPCRKit (AMV) ...
Embodiment 3
[0114] Example 3 Inhibition of SLC26A9 Gene Expression
[0115] 1. Cell culture (human rectal adenocarcinoma cell line SW480):
[0117] a. Take out the cell cryopreservation tube from the liquid nitrogen tank, immediately put the cell cryopreservation tube into warm water at 37°C, and shake it gently to melt it as soon as possible.
[0118] b. Take out the cryopreservation tube from the warm water at 37°C, suck out the cell suspension with a pipette, add 5ml of L-15 culture medium containing 10% bovine serum to the centrifuge tube, mix and centrifuge at 800rpm for 5min, remove the supernatant, Wash again with culture medium.
[0119] c. Add 5ml of L-15 culture solution containing 10% fetal bovine serum, inoculate in a 75ml culture bottle, and place at 37°C, 5% CO 2 cultured in a constant temperature incubator.
[0120] 2) Cell replacement
[0121] a. When the culture solution starts to turn yellow, take out the culture bottle from the incubator a...
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