Use of linc00266-1 RNA as a marker for solid tumors
A solid tumor, RT-PCR technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the lack of tumor monitoring indicators and other problems, achieve high mortality, increase positive rate, and short survival period effect
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Embodiment 1
[0044] RT-PCR reaction was used to detect the expression of LINC00266-1 gene in gastric cancer and liver cancer tissues.
[0045] The specific experimental plan is as follows:
[0046] 1. RNA extraction
[0047] 1) Tissue processing: Take about 10 mg of tissue and add 1 ml Trizol, homogenize with a homogenizer; centrifuge for 15 min at 12000 g, and take the supernatant.
[0048] 2) Add 200ul of chloroform to the supernatant, mix vigorously up and down for 3sec, and let stand for 3min.
[0049] 3) Centrifuge at 12,000 g for 15 minutes at 4°C. At this time, it can be seen that the lysate is divided into three layers: the upper layer is the water phase containing RNA; the middle layer is DNA, lipids, etc.; the lower layer is cell residues, proteins, polysaccharides, etc.
[0050] 4) Take the supernatant into a new EP tube; add an equal volume of isopropanol, mix well, let stand for 10 minutes, and then centrifuge at 12000g for 10 minutes at 4°C.
[0051] 5) Carefully remove th...
Embodiment 2
[0063] Q-PCR reaction was used to detect the expression of LINC00266-1 gene in gastric cancer and liver cancer tissues.
[0064] Q-PCR: GAPDH was used as an internal reference. Reaction system for Q-PCR: Add 10 μl to each reaction tube Premix Ex TaqTMII Buffer (2×), 0.8 μl forward primer, 0.8 μl reverse primer, 2 μl cDNA template prepared in Example 1, 0.4 μL ROX Reference Dye (50×), 0.2 μl Taq enzyme, add water to 20 μl . The Q-PCR reaction conditions are as follows:
[0065] ①95℃10min
[0066] ②95°C 15s
[0067] ③62℃1min
[0068] ④72°C 20s
[0069] ⑤go to②③④for 40 cycles
[0070] Primers used are listed below:
[0071]
[0072] The real-time fluorescent quantitative PCR results were analyzed in the Bio-Rad CFX Manager system software, and the amplification results were observed according to the fluorescence signal growth curve and the reaction critical cycle number (cycle threshold, Ct) given by the instrument. Using the 2-ΔΔCt value formula method to compare th...
Embodiment 3
[0074] RT-PCR reaction to detect the effect of siRNA silencing LINC00266-1 gene
[0075] (1) Digest the cells into a single-cell suspension, count, and use 5×10 5 The concentration of cells / well was inoculated into 6-well plates at 37°C, 5% CO 2 Incubate overnight in the incubator.
[0076] (2) Dissolve siALKBH1 in RNase-free deionized water to a final concentration of 20 μM, take 5 μl and dissolve in 500 μlopti-MEM, mix well, and let stand.
[0077] Wherein, the related siRNA sequence is as follows:
[0078] siLINC00266-1#1-sense:
[0079] 5′-CGUUUCACCUGCAGUUGAA TT -3' (SEQ ID NO: 7)
[0080] siLINC00266-1#1-anti-sense:
[0081] 5′-UUCAACUGCAGGUGAAACG TT -3' (SEQ ID NO: 8);
[0082] siLINC00266-1#2-sense:
[0083] 5′-CGUUUCACCUGCAGUUGAA TT -3' (SEQ ID NO: 9),
[0084] siLINC00266-1#2-anti-sense:
[0085] 5′-UUCAACUGCAGGUGAAACG TT -3' (SEQ ID NO: 10);
[0086] Negative control siRNA-sense:
[0087] 5′-GCACAAGCUGGAGUACAACUACA TT -3' (SEQ ID NO: 11),
[008...
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