"<h4>The concentration of MTT is 5mg/ml. Weigh MTT 0.5 g, dissolve in 100 ml of phosphate buffer solution (PBS) or culture medium without phenol red, with 0.22 μm filter membrane filtration to remove the bacteria in the solution, store at 4 ℃ and avoid light preservation. Containers are best wrapped in aluminum foil during preparation and preservation.</h4>"+

"<h4>HepG2 cell (3* 10⁴ /mL)s were seeded in 96-well plate according to 100 µL/ well, cultured at 37 ℃ and 5% CO2 constant temperature incubator.</h4>"+

"<h4>1. For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.</h4>"+

"<h4>2. Add 20 µL of the 12 mM MTT stock solution (prepared) to each well. Include a negative control of 20 µL of the MTT stock solution added to 100 µL of medium alone.</h4>"+

"<h4>3. Incubate at 37°C for 4 hours. At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.</h4>"+

"<h4>4. Add 150 µL of the DMSO solution to each well and mix thoroughly using the pipette.</h4>"+

"<h4>5. Incubate the microplate at 37°C for 10 min in a humidified chamber. Longer incubations will decrease the sensitivity of the assay.</h4>"+

"<h4>6. Mix each sample again using a pipette and read absorbance at 570 nm as detection wavelength and 630 nm as reference wavelength.</h4>"+

var qPCR = "<h2>The extraction of RNA</h2>"+

"<h3><i>A. Required reagents:</i></h3>"+

"<h3><i>D: Homogenization:</i></h3>"+

"<h4>Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of Transzol UP Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of Transzol UP Reagent added is based on the area of the culture dish (1 ml per 10 cm²) and not on the number of cells present. An insufficient amount of Transzol UP Reagent may result in DNA contamination of the isolated RNA.</h4>"+

"<h3><i>E. PHASE SEPERATION:</i></h3>"+

"<h4>Add 0.2 ml of chloroform per 1 ml of Transzol UP Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x.g for 15 minutes at 2 to 8℃. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of Transzol UP Reagent used for homogenization).</h4>"+

"<h3><i>F. RNA PRECIPITATION:</i></h3>"+

"<h4>Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of Transzol UP Reagent used for the initial homogenization. Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 2 to 4℃. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.</h4>"+

"<h3><i>G: RNA WASH:</i></h3>"+

"<h4>Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of Transzol UP Reagent used for the initial homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x.g for 5 minutes at 2 to 8℃. Repeat above washing procedure once. Remove all leftover ethanol.</h4>"+

"<h3><i>H. REDISSOLVING RNA:</i></h3>"+

"<h4>Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A₂₆₀/A₂₈₀ ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip.</h4>"+

"<h3><i>I. SPECTROPHOTOMETRIC ANALYSIS:</i></h3>"+

"<h4>Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). Using 10 μl microcuvette, take OD at 260 nm and 280 nm to determine sample concentration and purity. The A₂₆₀/A₂₈₀ ratio should be above 1.6. Apply the convention that 1 OD at 260 equals 40 µg /ml RNA.</h4>"+

"<h4>Use Vazyme miRNA 1st Strand cDNA Synthesis Kit (by stem-loop)</h4>"+

"<h4>Gently blow with a pipette to mix.</h4>"+

"<h4>Gently blow with a pipette to mix.</h4>"+

"<h4>Use Vazyme ChamQ Universal SYBR® qPCR Master Mix</h4><br>"+

"<h4>1.Grow cultured cells on chamber slides overnight, or add appropriate amount of cells to poly-L-lysine coated chamber slides and incubate at least 30 min at 37°C, at the time of fixation cells should be ~50% confluent. </h4>"+

"<h4>3.Fix cells by incubation with 4% Paraformaldehyde, in PBS for 15 min at room temperature. </h4>"+

"<h4>Block samples in 5% normal serum from same species as secondary antibody in 1% BSA/0.2% Triton X-100/PBS for 1 h at room temperature, or overnight at 4°C.</h4><br>"+

"<h4>8.Add 200 µl per well (8 wells) to the chamber slides and incubate 2 h at room temperature, or overnight at 4°C. </h4>"+

"<h4>13. Add DAPI solution in 1% BSA/0.05% Triton X -100/PBS, and incubate 15 min at room temperature.</h4>"+

"<h4>16. Take pictures under a fluorescence microscope</h4><br>";