Self-provided reagents
Chloroform, isopropanol, 70% ethanol (DEPC water configuration), Rnase Free H 2O.
Ⅰ Preparation before experiment
The key to RNA preparation is to inhibit RNA degrading enzymes in cells and prevent contamination of RNA degrading enzymes in the equipment and reagents used. Therefore, the following measures must be taken in the experiment: wear disposable clean gloves; use a special experimental bench for RNA operation; avoid talking during the operation, etc. The above methods can prevent the contamination of the experimenter's sweat and saliva by RNA degrading enzymes.
Cautions:
1. Try to use disposable plastic utensils. If glassware is used, it should be treated with 0.1% DEPC aqueous solution at 37°C for 12 hours before use, and then autoclaved at 120°C for 30 minutes to remove residual DEPC.
2. Reagents used for RNA experiments must be sterilized by dry heat (180°C, 60min) or DEPC water treatment related containers. The sterile water used must be treated with 0.1% DEPC and then autoclaved.
3. Reagents and sterile water for RNA experiments should be dedicated to avoid cross-contamination after mixing.
Ⅱ Experimental operation
Sample size and RNA yield
| Sample type |
Sample size |
RNA yield |
| Human whole blood |
250 μl |
3~10 μg |
| Leukocyte |
1x106 |
10~20 μg |
| Cells |
1x106 |
8~15 μg |
| Tissues such as muscle/brain |
25 mg |
10~25 μg |
| Liver |
25 mg |
50~100 μg |
TRIzol LS usage instructions
|
Liquid samples: Anticoagulated whole blood, Aerum, Virus liquid |
Suspension cells, Yeast, Bacteria |
Animal and Plant tissue |
Adherent cells |
| 1. Sample pretreatment |
For solid samples such as feces, the samples can be resuspended in PBS, homogenized, centrifuged at 3000 rpm for 5 minutes, and the supernatant is taken as the virus liquid sample. Other samples can be used directly. |
If the cell content in the sample is low, the cells need to be precipitated by centrifugation and then resuspended in 250 μl sterile water before proceeding to the following operations. |
Transfer the sample to a mortar pre-cooled with liquid nitrogen, grind the tissue with a pestle, and continuously add liquid nitrogen in the meantime until it is ground into a powder. |
Pour out the culture solution from the cultured cells every 10 cm2 and wash them with PBS once to remove as much excess solution as possible. |
| 2. Add TRIzol LS |
Take 250 μl liquid sample and add it to a 1.5ml EP tube containing 750 μl TRIzol-LS. |
Add 750μl TRIzol-LS to 250μl liquid sample. |
Add the ground tissue to a 1.5ml EP tube containing 750 μl TRIzol-LS. |
Add 750 μl TRIzol-LS to evenly distribute the lysate on the cell surface, then use a pipette to blow the cells off and transfer them to a 1.5ml EP tube. |
| 3. Lysed sample |
After adding TRIzol-LS, immediately turn the wrist upside down until the cells and tissue powder are evenly dispersed without lumps. Leave it at room temperature for 5 minutes to completely separate the nucleic acid-protein complexes. |
| 4. Add water |
No additional water is required for liquid samples. |
Add 250 μl Rnase Free H2O, mix well, and let stand for 2 minutes. |
| 5. Add Chloroform |
Add 200 μl chloroform, shake vigorously with the wrist for 15 seconds, and leave it at room temperature for 2 minutes. |
| 6. Centrifugal layering |
Centrifuge at 13,000 rpm for 10 minutes, and transfer 600 μl of colorless supernatant to a new 1.5 ml EP tube. |
| 7. Add isopropanol |
Add 600 μl of isopropanol to the above 600 μl of supernatant, turn it upside down several times vigorously with the wrist, and place it at -20°C for 5 minutes. |
| 8. Centrifugation of total RNA |
Centrifuge at 13,000 rpm for 10 minutes, carefully discard the supernatant, and save the bottom total RNA pellet. |
| 9. Rinse total RNA |
Add 1 ml of 70% ethanol to each tube of the pellet, turn it upside down several times, centrifuge at 13,000 rpm for 5 minutes, carefully discard the supernatant, and save the bottom RNA pellet. |
| 10. Repeat the rinse one more time |
Repeat step 9 to wash again. |
| 11. Volatile residual ethanol |
Pour off the washing solution, centrifuge again for a short time for 10 seconds, absorb the remaining washing solution with a 10 μl tip, and place it at room temperature to evaporate the ethanol (~20min). |
| 12. Dissolve total RNA |
Add 20-100 μl TE Buffer or RNase Free H2O to each tube to dissolve total RNA. |
Common problems
1. Low extraction rate. Possible reasons: (a. Sample lysis or homogenization is not complete; b. RNA precipitation is not completely dissolved)
2. A260/A280<1.65. Possible reasons: (a. When measuring the absorbance, the RNA sample was not dissolved in water, but dissolved in TE; b. The amount of tissue added when the sample was homogenized was too much; c. After stratification, the supernatant was less than 500μl; d. The organic phase was mixed when the water phase was absorbed)
3. Excessive DNA contamination. Possible reasons: (a. The amount of reagents added during sample homogenization is too small or the amount of tissue is too much; b. The sample contains organic solvents) Solution: using this reagent usually genomic DNA contamination content <0.1ng/μl, if it is necessary to remove DNA contamination completely, please use Rnase Free DNase I to digest and remove genomic DNA contamination. If you use the Gold Medal cDNA First Strand Reverse Transcription Kit there is no need to digest to remove genomic DNA contamination in advance The kit contains reagents to remove genomic DNA contamination.
