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This product RNA extraction solution is a reagent used for total RNA extraction from cells or tissues. It uses guanidine isothiocyanate and other substances to rapidly lyse cells to release RNA and extract it into phenol. At the same time, it contains RNase inhibitors to prevent RNA degradation and maintain RNA integrity.
RNA extraction solution
Wet ice bag transportation; Stored at 4℃ temperature, valid for 12 months.
Self-prepared LF, isopropyl alcohol, DEPC water, 75% ethanol (DEPC water preparation).
1. Lysis of a cell or tissue sample
Adhered cells: remove cell culture medium, clean cells with appropriate amount of pre-cooled PBS, and blot out PBS as much as possible. Add RNA extract to the well plate according to the amount of 1 mL RNA extract per 6-well plate and 0.5mL RNA extract per 12-well plate, shake gently to make the cells at the bottom of the plate fully contact the RNA extract, blow the cells with pipette gun to make the cells lyse, then transfer to the centrifuge tube, and let stand at room temperature for 5 min.
Suspend cells: Cells were collected by centrifugation, and the supernatant was removed as far as possible. 1mL RNA extract was added to every 1-10x106 cells, and the cells were blown several times with pipette gun to cleavage, then the cells were left standing at room temperature for 5min.
Tissue sample: cut the tissue into small pieces and put them into a pre-cooled homogenizer. Add 1mL RNA extract for every 100mg of tissue and homogenize until no tissue pieces are visible. The tissue fragments were separated by centrifugation at 12000g at 4℃ for 10min, and the supernatant was transferred to a new centrifuge tube.
2. RNA extraction: 380ul LF was added to each 1mL RNA extract, and then the centrifuge tube was shaken violently for 15s by eddy mixing or upside-down, and left for 3min at room temperature.
3. Centrifugation: centrifuge at 12000 g at 4℃ for 15 min, carefully transfer the upper colorless aqueous phase to the new centrifuge tube, and absorb 500-550ul per ml of extract.
4. Precipitated RNA: 550ul isopropanol was added to the aqueous phase extract tube collected above, mixed slightly upside down for several times, and precipitated at -20℃ for 15min.
5. Centrifugation: centrifuged at 4℃ for 10min at 12000g, white precipitation was visible at the bottom of the tube, which was total RNA, and supernatant was discarded.
6. Washing: Add 1 mL 75% ethanol and mix it upside down. After centrifugation at 12000g for 5min at 4℃, the supernatant was discarded and then centrifuged briefly at a high speed (5000g for 3-5s). The liquid was carefully sucked out with a micro suction head.
7. RNA dissolution: The centrifuge tube with RNA precipitation was left open for 3-5min. After the RNA was slightly dried, 20ul DEPC water was added to make the RNA fully dissolved, which could be used for concentration detection and follow-up experiments. Be careful not to dry the RNA too much, otherwise it will be difficult to dissolve and will affect the A260/280 ratio.
1. All pipette tips and centrifuge tubes shall be free of RNase contamination. You can purchase sterile enzyme-free consumables from our company.
2. This reagent contains guanidine isothiocyanate and resteamed phenol. Avoid contact with skin or inhalation.
3. Please wear lab clothes and disposable gloves.