RNA is an intermediate product of gene expression and also the genetic material of RNA viruses. The manipulation of RNA plays an important role in molecular biology. Highly pure and complete RNA is necessary for many molecular biology experiments. The success or failure of experiments such as Northern hybridization, cDNA synthesis and in vitro translation depends to a large extent on the quality of RNA. The RNA in plant cells is mainly rRNA (80% - 85%), tRNA and small RNA (10% - 15%) and mRNA (1% - 5%). rRNA is the most abundant and consists of 28S, 18S and 5S.
Principle
Trizol reagent is a single-phase, rapid extraction of total RNA formulated from phenol and guanidinium thiocyanate. During the homogenization and lysis process, it can break cells, degrade proteins and other components, separate proteins from nucleic acids, and inactivate RNase, while maintaining the integrity of RNA. After chloroform extraction and centrifugation, the RNA is in the aqueous phase. The aqueous phase is transferred to a tube and the RNA is precipitated with isopropanol.
Procedures
- Autoclave the pipette tip, mortar, pestle, and PCR tube at 121°C for 1 h.
- Take about 100 mg of fresh tobacco leaves, quickly freeze the biological material in liquid nitrogen, and then grind the tissue in a mortar using liquid nitrogen grinding. If you do not grind it immediately, you can first store the liquid nitrogen-frozen plant tissue in a -80°C refrigerator.
- Transfer the ground and broken tissue to a tube, and immediately add 1 mL Trizol reagent, shake vigorously for 15 s, and then leave it at room temperature for 5 min.
- Centrifuge at 12,000 g for 5 minutes at 4°C, and transfer the supernatant to another clean tube.
- Add 0.2 mL chloroform to the tube, shake vigorously for 15 s, and place at room temperature for 3 min.
- Centrifuge at 12,000 g for 15 minutes at 4°C and transfer the supernatant to another clean tube.
- Add 0.5 mL isopropyl alcohol to the tube, gently invert the tube 5 times, and leave it at room temperature for 5 minutes.
- Centrifuge at 12,000 g for 5 minutes at 4°C and discard the supernatant.
- Add 1 mL of 75% ethanol for washing, gently invert the tube 5 times, centrifuge at 12,000 g for 3 min at 4°C, and discard the supernatant.
- Let the precipitated RNA dry naturally at room temperature for about 5 minutes.
- Add 30 µL DEPC water and incubate at 60°C for 5 minutes to dissolve the RNA precipitate.
- Dilute the dissolved RNA 50 times and measure the RNA concentration and OD260/280 with a spectrophotometer.
- OD260/OD280 should be 1.7-2.1. Below 1.7 indicates protein and phenol contamination. OD260/OD230 should be greater than 2.0. If it is lower than this value, it means there is contamination by small molecules such as sugar, salt, and guanidine. A solution with an OD260 value of 1 contains approximately 40 µg/mL RNA, so the concentration of total RNA (µg/mL) = OD260 value × 40 µg/mL × dilution factor.
- Take 1 µL of the lysate and conduct 1% agarose gel electrophoresis. Electrophoresis was performed at 100 V for 15 min, and then the results were observed under UV light.
Note:
- All utensils need to be treated with 0.05% DEPC (ethylene pyrocarbonate) solution for more than 30 min before the experiment. The solvent water used in the experiment also needs to add 0.05% DEPC to inhibit the activity of possible remaining RNase.
- Trizol, DEPC, etc. are toxic and may cause injury in contact with skin. Wear gloves during operation and operate under ventilated conditions.
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