Animal tissues and cells are the most commonly used experimental materials in molecular biology research, a typical mammalian cell contains about 105 ug RNA, which is mainly rRNA, tRNA and other small molecules in the nucleus, as well as a small number of mRNA. general experiments only need to extract the total RNA of the tissues or cells, but some experiments that have special requirements require specific RNA components from the total RNA isolation. However, for some special experiments, it is necessary to isolate specific RNA components from total RNA. This section describes common methods for extracting total RNA, rRNA and mRNA from animal tissues, cells and specimens.
Operation method
TRlzol reagent for one-step RNA isolation
Principle
TRIzol Reagent is a ready-to-use reagent for the isolation of total RNA from cells and tissues that contains a monophasic solution of phenol and guanidinium thiocyanate, a further refinement of Chomczynski and Sacchi's method for further RNA isolation. TRIzol reagent not only lyses cells but also maintains RNA integrity during homogenization or sample lysis. After addition of chloroform and centrifugation, the solution is divided into an aqueous phase and an organic phase, with the majority of the RNA retained in the aqueous phase. After the RNA has been transferred to the aqueous phase, the RNA is precipitated with isonitol, and after removal of the aqueous phase, the DNA and proteins in the sample are obtained by sequential precipitation. DNA is obtained in the intermediate phase by ethanol precipitation, and proteins are obtained in the organic phase by isopropanol precipitation. This technique is suitable for the isolation of RNA from small amounts of tissue (50 to l00 mg) and cells (5X104) as well as large amounts of tissue (≥lg) and cells (5X107) from humans, animals, plants, and bacteria, and is very effective, with the entire process being completed in less than an hour. Total RNA isolated with TRIzol is free of protein and DNA contamination and can be used for Morrhernblot analysis, spot hybridization, pdy(A) selection, in vitro translation, RNase protection analysis, molecular cloning, etc. TRIzol is easy to isolate various RNAs of different molecular masses, e.g., RNA isolated from rat liver, agarose electrophoresis at 7kb and 15kb shows the same results. For example, RNA isolated from rat liver, after agarose electrophoresis, showed different high molecular mass RNA bands at 7kb and 15kb, with the two major ribosomal RNA bands at ~5kb (28S) and ~2kb (18S); the low molecular mass RNA was 0.1-0.3 kb (tRNA, 5SRNA). Isolated RNA260/280= 1.6~1.8. The yield of RNA per mg of tissue is: liver and spleen, 6~10 ug, kidney 3~4 ug, skeletal muscle and brain 1~5 ug, placenta 1~4 ug. The amount of RNA obtained from 1X106 cells is: epithelial cells 8~15 ug, fibroblasts 5~7 ug. There are now several TRIzol analogs available. Available.
Materials and Instruments
Chloroform Isopropyl alcohol Ethanol RNase-free water SDS solution TRIzol reagent Move I. Materials and equipment Caveat 1) Low RNA yield: This may be due to the following reasons: the sample was not homogenized or lysed completely; the RMA precipitate was not completely solubilized.2)A260/280＜1.65 This may be due to the following reasons: the sample is too small; after homogenization, the specimen is not left at room temperature and the aqueous phase is contaminated with phenol; the RNA precipitation is not completely dissolved.3) RNA degradation: the possible causes are as follows: tissues removed from animals are not processed or frozen immediately; samples for RNA isolation are stored at -5 to 20°C instead of 60 to 70°C; cells are digested by trypsin; RNase is contained in solutions or tubes; formaldehyde used for agarose gel electrophoresis is at a pH ＜3.4) DNA contamination: This may be due to the following reasons: too few samples; samples used for isolation contain organic solvents such as ethanol, DMSO) though buffers or alkaline solutions.5) Proteoglycan and polysaccharide contamination: The following modified RNA precipitation method (step 3), removes these contaminants from the isolated RNA. Add 0.25 ml of isopropanol per ml of TRIzolml to the aqueous phase, plus 0.25 ml of seolite precipitation solution (0.8 mol/L sodium citrate 1.2 mol/L NaCl), mix, centrifuge, and proceed as before. This effectively precipitates the RNA while the proteoglycans and polysaccharides remain in the liquid phase.6) From a small number of samples (cells ≤ 1046) To extract RMA from a small amount of sample (cells ≤ 10 4 or tissue ≤ 10 mg), add 0.8 ml of TRIzol reagent, homogenize, add chloroform, and perform RNA isolation as in step 2. Before precipitating the RNA with isopropanol, add 5 to 10 µg of RNase-free glycogen.7) If the sample contains a high concentration of proteins, polysaccharides, or extracellular substances, the muscle should be isolated. polysaccharides or extracellular substances, muscle, adipose tissue, plant stem mass, additional isolation is required. After homogenization, centrifuge the sample at 2 to 12,000 g for 10 mm to remove insoluble material. The resulting precipitate contains extracellular membranes, polysaccharides, and high-molecular-quantity DNA, and the supernatant contains RNA. samples from adipose tissues, in which the fat accumulates in the upper layer, can be removed. Transfer the homogenate to a new tube and add chloroform for liquid phase separation as described above.8) After homogenization and before addition of chloroform, the samples can be stored at -60 to -70°C for at least one month. RNA precipitate can be stored in 75% ethanol at 2 to 8°C for at least one week, and at -5 to 20°C for at least one year.9) If the centrifugation speed is 2600 g in steps 2) and 3), increase the time to 20-30 min.10) TRIzol reagent is toxic, so if it comes into contact with the skin, wash it well with water. If you feel unwell, go to the hospital as soon as possible.TRIzol reagent should be stored at 2~8℃, if stored at room temperature, the expiration date is 12 months. For more product details, please visit Aladdin Scientific website.
1) Chloroform
2)Isopropyl alcohol
3)75% ethanol (prepared with DEPC water)
4)RNase-free water (add water to RNase-free glass vials, add DEPC at a concentration of 0.1 % ( V /V) overnight , sterilize under pressure)
5) 0.5% SDS solution (must be DEPC treated autoclaved water preparation )
6) TRIzol reagent
Operation method
1 . Homogenization
1 ) Tissue: For every 50~l0 mg of tissue, add 1 ml of TRIzol reagent and homogenize with a glass-Teflcm or electric homogenizer. The sample volume should not exceed 10% of the TRIzol reagent.
2 ) Cells in monolayer culture: Add 1ml of TRIzol Reagent to a 3.5cm dish - pipette the cells several times and lysed them directly. The amount of TRIzol reagent to be added depends on the size of the dish (1ml / 10cm2 ) and not on the amount of cells. insufficient amount of TRIzol reagent makes the isolated RNA susceptible to DNA contamination.
3) Suspension of cells: collect the cells by centrifugation, add TRIZol Reagent and repeatedly blow the plants to lyse the cells. For animals, plants and yeast, use lml of TRIzol reagent for every 5x106~10x106 cells. Do not wash the cells before adding TRIzol Reagent as this may increase RNA degradation. Shade and yeast cells can be lysed with a homogenizer.
2 . Separation of phases
1 ) After homogenization, leave at 15~30 ℃ for 5min to ensure complete separation of nucleoprotein complexes.
2 ) Add 0.2m l chloroform/lm l TRIzol reagent. Cover the tube and let stand for 2-3 min at 15-30°C with vigorous hand shaking for 15 s. Centrifuge at 2-8°C for 10 mm at less than 12,000 g. After centrifugation, the mixture is divided into three phases: the lowest red phenol-chloroform phase, the middle phase, and the upper aqueous phase & the majority of the RNA is retained in the aqueous phase, which is used for 60% of the volume of homogenized TRIzol reagent.
3. RNA precipitation
1 ) Transfer the aqueous phase to a new tube (keep the organic phase if you want to isolate DNA or proteins), add isopropanol and mix well to precipitate the RNA. add 0.5 ml of isopropanol for every 1 ml of TRlizol reagent, and let it stand for 10 min at 15~30°C. 2) Add the aqueous phase to a new tube.
2) Centrifuge at 2~8℃ for 10min at less than 12000 g. Before centrifugation, RNA precipitation is often not visible, but after centrifugation, RNA forms a gelatinous pellet. Sedimentation occurs at the bottom and on the wall of the tube.
4. RNA washing
1) Remove the supernatant and wash the RNA precipitate once with 75% ethanol, add at least 1ml of 75% ethanol for every 1ml of TRIzol reagent and mix well, then centrifuge the sample at 7500g at 2-8°C for 5min.
2 ) Discard the supernatant
5. RNA solubilization
1) Air dry the RNA precipitate for 5~l 0 min. Do not centrifuge the RNA under vacuum, and be careful not to dry the RNA completely, as this will greatly reduce its solubility. If the RNA is not completely solubilized, its A260/280 is <1.6%.
2 ) Add RNase-free water or 0.5% SDS solution and blow repeatedly to dissolve the RNAg If the RNA is difficult to dissolve, dissolve it at 55~60℃ for 10min.