First, the basic principles of separation and purification Regulation of gene expression and often isolated from tissues and cells and purification of RNA . RNA quality often affect the level of cDNA library, RT-PCR and Northern Blot the success of molecular biology experiments. Trizol is a new total RNA extraction reagent containing guanidine isothiocyanate and other substances, can be quickly broken cells, inhibit cell release of nucleic acid enzymes.
Second, the user should prepare the reagents and materials for ethanol, chloroform, Glycogen (may need), 1.5ml Eppendorf tube (RNase-free), ips (RNase-free)
Third, the preparation RNase enzyme is very stable, leading to RNA degradation is the most important material. In extreme conditions it can be temporarily inactivated, but the limiting factor in the rapid refolding after removal. With conventional high-temperature high-pressure steam sterilization methods and protein inhibitors can not be RNase completely inactivated. It is widely present in human skin, therefore, prepared with RNA molecular biologyrelated experiments, must wear gloves. RNase is another source of pollution is taking dispenser, according to the manufacturer take the fluid requirements of the pipetting device for processing. Under normal circumstances the use of prepared with DEPC scrub take 70% ethanol solution's internal and external, to achieve the basic requirements.
RNase-free items to take, when to wear gloves.
1. the possible use of material handling products, sterile, disposable plastic products, has been labeled RNase-Free plastic products, Kaifeng used without re-treatment is usually not necessary. Plastic products for domestic, in principle, must be handled before use. Processing steps are as follows :
- into a glass beaker of deionized water, add DEPC. DEPC to a final concentration of 0.1%. Note: DEPC Is highly toxic, highly reactive, be careful in the use of fume hoods.
- processing of plastic products can be placed in a high-temperature sterilization container, into the DEPC solution, so that all parts are plastic immersed into the solution.
- treatment at room temperature overnight in a fume hood.
- Carefully pour the DEPC solution to the waste bottle, sealed with aluminum foil over the plastic has been DEPC water treatment products Beaker, high-temperature high-pressure steam sterilization at least 30 minutes.
- bake oven with a copy to the appropriate temperature drying. Placed in a clean place back.
Fourth, from the organization in total RNA extracted
1) liquid nitrogen grinding, tissue directly into the mortar, add a small amount of liquid nitrogen, quickly ground, to be soft tissue, together with a small amount of liquid nitrogen, then ground, so three times, according to 50-100mg tissue / ml Trizol adding Trizol , into the centrifuge tube for step 2.
2) homogenizer: tissue samples by 50-100mg/mlTrizol adding Trizol. In addition, the tissue volume can not exceed the Trizol 10% of the volume, otherwise the effect will be bad homogenate, fully homogenized with an electric homogenizer about 1-2 minutes.
1) liquid nitrogen grinding, tissue directly into the mortar, add a small amount of liquid nitrogen, quickly ground, to be soft tissue, together with a small amount of liquid nitrogen, then ground, so three times, according to 50-100mg tissue / ml Trizol adding Trizol , into the centrifuge tube for step 2.
2) homogenizer: tissue samples by 50-100mg/mlTrizol adding Trizol. In addition, the tissue volume can not exceed the Trizol 10% of the volume, otherwise the effect will be bad homogenate, fully homogenized with an electric homogenizer about 1-2 minutes.
Fifth, from the cell total RNA
1) to develop adherent cells: do not need to digest, can be directly carried out using Trizol digestion, pyrolysis, Trizol volume by 10cm2/ml The proportion of added.
2) suspension cells can be collected directly, pyrolysis, each 1ml Trizol lysis 5 × 106 can be animal, plant or yeast cells, or 107 bacterial cells.
Six steps
1, cells or tissues after Trizol added, at room temperature for 5min, to make it fully cracking. Note: At this point -70 ℃ can be placed in long-term.
2,12,000 rpm centrifuge 5min, discard precipitate.
3, according to 200ul chloroform / ml Trizol added chloroform, Shakers at room temperature after 15min. Note: Disabling the vortex oscillator, In order to avoid genomic DNA.
4,4 ℃ 12,000 g centrifugation 15min.
1, cells or tissues after Trizol added, at room temperature for 5min, to make it fully cracking. Note: At this point -70 ℃ can be placed in long-term.
2,12,000 rpm centrifuge 5min, discard precipitate.
3, according to 200ul chloroform / ml Trizol added chloroform, Shakers at room temperature after 15min. Note: Disabling the vortex oscillator, In order to avoid genomic DNA.
4,4 ℃ 12,000 g centrifugation 15min.
5, draw the upper aqueous phase to another centrifuge tube. Note: Do not learn the middle of the interface; if at the same extracted DNA and protein, retains the lower phenol phase stored in 4 ℃ refrigerator, if only to mention RNA, then discard the lower phenol phase.
6, according to the 0.5ml isopropanol /ml Trizol added isopropyl alcohol mixing at room temperature for 5-10min. 7,4 ℃ 12,000 g centrifugation 10min, supernatant, RNA Shen in the bottom of the tube.
8, according to 1ml 75% ethanol / ml Trizol adding 75% ethanol, a mild oscillation centrifuge tube, suspended sediment. 9, 4 ℃ 8,000 g centrifugation 5min, supernatant as possible.
10, room temperature drying or vacuum drying 5-10min. Note: RNA samples not to be too dry, it would be difficult to dissolve.
11, available 50ul H2O, TE buffer, or 0.5% SDS dissolved RNA samples ,55-60 ℃ ,5-10min. Note: H2O, TE or 0.5% SDS using DEPC treatment and high pressure are required.
12, the measured OD values of quantitative RNA concentration. Note: This method RNA A260/A280 value between 1.6-1.8; yield estimates: tissue samples: (UG RNA / mg组织) 1-10ug, cultured cells (UG RNA / 106 Cell) :5-15ug. Note: Tissue or cell volume is too small, the amount of discretion to reduce the Trizol; excessive amount of tissue or cells, can cause DNA to RNA Pollution; protein, fat or carbohydrate tissue, muscle tissue or plant tissue such as block, tissue or liquid nitrogen was ground to be 4 ℃ 12,000 g centrifugation 10min to remove insoluble material, then the following operations, if the top level there is fat material, it is also to be removed; Hot days mentioned RNA, with the gloves is a must, the hand is the main source of RNase; tissue grinding with liquid nitrogen, the best, If there is no liquid nitrogen or electric homogenizer can be used instead of manual homogenizer, tissue at this time should not be too large, and the need to first base with ophthalmic scissors to organization base was cut, and then fully ground.
6, according to the 0.5ml isopropanol /ml Trizol added isopropyl alcohol mixing at room temperature for 5-10min. 7,4 ℃ 12,000 g centrifugation 10min, supernatant, RNA Shen in the bottom of the tube.
8, according to 1ml 75% ethanol / ml Trizol adding 75% ethanol, a mild oscillation centrifuge tube, suspended sediment. 9, 4 ℃ 8,000 g centrifugation 5min, supernatant as possible.
10, room temperature drying or vacuum drying 5-10min. Note: RNA samples not to be too dry, it would be difficult to dissolve.
11, available 50ul H2O, TE buffer, or 0.5% SDS dissolved RNA samples ,55-60 ℃ ,5-10min. Note: H2O, TE or 0.5% SDS using DEPC treatment and high pressure are required.
12, the measured OD values of quantitative RNA concentration. Note: This method RNA A260/A280 value between 1.6-1.8; yield estimates: tissue samples: (UG RNA / mg组织) 1-10ug, cultured cells (UG RNA / 106 Cell) :5-15ug. Note: Tissue or cell volume is too small, the amount of discretion to reduce the Trizol; excessive amount of tissue or cells, can cause DNA to RNA Pollution; protein, fat or carbohydrate tissue, muscle tissue or plant tissue such as block, tissue or liquid nitrogen was ground to be 4 ℃ 12,000 g centrifugation 10min to remove insoluble material, then the following operations, if the top level there is fat material, it is also to be removed; Hot days mentioned RNA, with the gloves is a must, the hand is the main source of RNase; tissue grinding with liquid nitrogen, the best, If there is no liquid nitrogen or electric homogenizer can be used instead of manual homogenizer, tissue at this time should not be too large, and the need to first base with ophthalmic scissors to organization base was cut, and then fully ground.