New application of hrnbp3 gene and its expression product in diseases
A gene and expression level technology, applied to the HRNBP3 gene and its expression products in the field of diagnosis and treatment of rectal adenocarcinoma, can solve problems such as tumor metastasis
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Embodiment 1
[0057] Example 1 Screening for gene markers associated with rectal adenocarcinoma
[0058] 1. Collection of samples
[0059]8 cases of rectal adenocarcinoma paracancerous tissues and rectal adenocarcinoma tissue samples were collected, and all the above samples were obtained with the consent of the organizational ethics committee.
[0060] 2. Preparation of RNA samples (operated using QIAGEN tissue RNA extraction kit)
[0061] 1) Tissue extraction
[0062] In a clean area with less RNase interference, use a mortar containing an appropriate amount of liquid nitrogen to weigh the isolated intervertebral disc nucleus pulposus tissue sample, grind it to powder with a pestle, and then transfer the sample to a RNase-free 2ml centrifuge tube middle. Add 300 μl of lysate, place in a homogenizer, grind thoroughly for 5 minutes, centrifuge at 12,000 g at 4°C for 10 minutes, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 600 μl of RNase-free water, mix with a vortex...
Embodiment 2
[0080] Example 2 QPCR sequencing to verify the differential expression of the HRNBP3 gene
[0081] 1. According to the detection results of high-throughput sequencing, the HNBP3 gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 70 cases of rectal adenocarcinoma paracancerous tissues and 70 cases of rectal adenocarcinoma tissues were selected.
[0082] 2. RNA extraction The steps are the same as in Example 1.
[0083] 3. Reverse transcription: use the reverse transcription kit of TAKARA company to operate. Specific steps are as follows:
[0084] (1) Take 1 μg of total RNA for reverse transcription, add 2 μl of Oligo(dT), and mix well. Immediately after 5 min in water bath at 70°C, ice bath for 2 min.
[0085] (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 wate...
Embodiment 3
[0106] Inhibition of embodiment 3HRNBP3 gene expression
[0107] 1. Cell culture: human rectal adenocarcinoma cell line HRC-99, cultured in RPMI1640 medium containing 10% calf serum and 1% P / S at 37°C, 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.
[0108] 2. siRNA design
[0109] siRNA sequences against HNBP3:
[0110] siRNA1-HRNBP3:
[0111] The sense strand is 5'-AGUUACAAACCCAAAACCCUU-3' (SEQ ID NO.7),
[0112] The antisense strand is 5'-GGGUUUUGGGUUUGUAACUUU-3' (SEQ ID NO.8);
[0113] siRNA2-HRNBP3:
[0114] The sense strand is 5'-AGUUUCAAAAGUUACAAACCC-3'(SEQ ID NO.9),
[0115] The antisense strand is 5'-GUUUGUAACUUUUGAAACUAG-3' (SEQ ID NO.10);
[0116] siRNA3-HRNBP3:
[0117] The sense strand is 5'-AUUAUUGACCUCAAUUUUCCG-3' (SEQ ID NO.11),
[0118] The antisense strand is 5'-GAAAAUUGAGGUCAAUAAUGC-3' (SEQ ID NO.12);
[0119] Nega...
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