Long non-coding RNA (Ribonucleic Acid) ENST 00000508435 gene and purpose thereof
A long-chain non-coding, useful technology, applied in the field of biomedicine, can solve the problems of lncRNA expression, function and its regulatory mechanism that have not yet been seen, and achieve the effect of promoting breast cancer cell migration and preventing breast cancer metastasis.
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Embodiment 1
[0039] Embodiment 1.RACE experiment
[0040] (1) 5'RACE experiment:
[0041] ①Design at least two antisense gene-specific primers with enzyme cleavage sites, gene-specific primer SP1 is used to reverse transcribe RNA into cDNA, SP2 is located upstream of SP1 for PCR amplification reaction (SP3 is located upstream of SP2 (for the second round of PCR amplification reaction); ②extraction of total cellular RNA; ③synthesis of cDNA with SP1; ④purification of cDNA; ⑤add polyA tail to the purified cDNA with dATP and terminal transferase; cDNA as a template, SP2 and Oligo dT-Anchor Primer as primers for PCR reaction (use the amplified product as a template, SP3 and Oligo dT-AnchorPrimer as primers for the second round of PCR reaction); ⑦The product is recovered by DNA electrophoresis gel, Connected into the pcDNA3.1 vector to form a recombinant plasmid, and sequenced to obtain the 5' end sequence;
[0042] (2) 3'RACE experiment:
[0043] ①Design a forward primer gene-specific forwar...
Embodiment 2
[0044] Example 2. Transwell chamber cell migration experiment
[0045] (1) Trypsinize the cells after interfering or transiently overexpressing lncRNA ENST00000508435 for 48 hours. After the digestion is complete, centrifuge to discard the liquid, resuspend the cells in serum-free medium, and count;
[0046] (2) Add 500 μl of culture medium containing 10% FBS to the lower chamber of the 24-well plate Transwell, add 200 μl of serum-free cell suspension to the upper chamber, and the number of cells is 0.5×10 5 , inoculate 3 duplicate wells, pay special attention, there should be no air bubbles between the lower medium and the small chamber, if air bubbles occur, lift the small chamber again, remove the air bubbles and then put the small chamber back into the culture plate;
[0047] (3) After 24 hours, suck out the liquid in the culture plate, wash gently with PBS 3 times, and fix with methanol for 30 minutes;
[0048] (4) Add 500 μl 0.1% crystal violet to each well of the cultu...
Embodiment 3
[0052] Example 3. Cell Migration Scratch Experiment
[0053] Basic principle of the experiment: The cell scratch method can simply measure the ability of cell migration and repair. In the cell culture dish, use a micropipette to draw a straight line in the cell growth area, set observation points at different times, and observe whether the surrounding cells repair to the scratch area. This was used to judge the ability of cell migration and growth.
[0054] Experimental steps:
[0055] (1) After interfering or overexpressing lncRNA ENST0000050843548h in BT549 cells or MDA-MB-231 cells respectively, use a pipette tip to scratch vertically to the plane of the culture plate, and use a ruler to scratch. When scratching, pay attention to the strength used between different wells The same, so as not to draw straight lines with different widths, and at the same time, do not use excessive force, so as not to scratch the bottom membrane of the culture plate and affect the growth of ce...
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