This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be completed simultaneously within 1 hour. This product does not require the ultra centrifugation step of CsCl purification and LiCl or ethanol precipitation. It does not contain toxic solvents such as phenol or chloroform. The purified RNA effectively removes enzyme inhibitors and pollutants such as heme and heparin. It can be directly used in various molecular biology routine experiments, such as RT-PCR, Northern Blot, Dot Blot, in vitro translation, and so on.
Self prepared reagents: β- Mercaptoethanol, 70% ethanol (prepared with water without RNase), anhydrous ethanol.
| R666034 |
Component |
50 T |
Storage |
| R666034A |
Buffer RBL (10×) |
60 mL |
RT |
| R666034B |
Buffer RL |
35 mL |
RT |
| R666034C |
Buffer RW1 |
40 mL |
RT |
| R666034D |
Buffer RW2 (concentrate) |
11 mL |
RT |
| R666034E |
RNase-Free Water |
10 mL |
RT |
| R666034F |
Spin Columns FL with Collection Tubes |
50 sets |
RT |
| R666034G |
Spin Columns RM with Collection Tubes |
50 sets |
RT |
| R666034H |
RNase-Free Centrifuge Tubes (1.5 mL) |
50 EA |
RT |
|
Preparation and important precautions before the experiment
To prevent RNase pollution, attention should be paid to the following aspects:
1) Use RNase free plastic products and gun heads to avoid cross contamination.
2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.
3) Prepare the solution using water without RNase.
4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
2. The sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.
3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be dissolved again in a 56 ℃ water bath. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL μ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.
4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.
5. This reagent kit cannot be used for RNA extraction from frozen blood samples with anticoagulants added.
6.10 × Buffer RBL needs to be diluted 10 times with water without RNase before use, and then stored at 2-8 ℃ after dilution.
7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.
8. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.
Operation steps
1. Add 5 times the volume of 1 x Buffer RBL to fresh anticoagulant whole blood samples of 0.5-1.5 ml (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex or invert and mix well. Incubate on ice for 10-15 minutes, mix twice during the incubation process.
Attention: During the incubation process, the cloudy suspension will become transparent, indicating that red blood cells have been lysed. If necessary, the incubation time can be extended to 20 minutes.
2.
Centrifuge at 4 ℃, 2100 rpm (~400 × g) for 10 minutes, and carefully discard the supernatant.
3. Add 2 times the volume of the blood sample to the above precipitate with 1 x Buffer RBL (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex, and resuspend the precipitate thoroughly.
4.
Centrifuge at 4 ℃ and 2100 rpm for 10 minutes, carefully and thoroughly remove the supernatant.
Note: This step must completely remove the supernatant, otherwise it will affect the lysis and lead to a decrease in RNA production.
5. Add Buffer RL to the precipitate (check if it has been added before use β- Mercaptoethanol, 0.5-1.5 ml of blood sample added to 600 μ L Buffer RL, or less than 0.5 ml of blood sample added to 350 μ L Buffer RL, mix well.
6. Transfer the obtained liquid to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, collect the filtrate, and discard the filter column.
7. Add 1 volume (600) to the obtained filtrate μ L or 350 μ l) Mix 70% ethanol (prepared without RNase water) well.
Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.
8. Add all the solution obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
9. Add 700 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 9 with the following steps.
1) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
2) Preparation of DNase I mixture: Take 70 μ Reaction Buffer and 10 μ L DNase I storage solution, gently mix and prepare to a final volume of 80 μ The reaction solution of L.
Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.
1) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
2) Preparation of DNase I mixture: Take 70 μ Reaction Buffer and 10 μ L DNase I storage solution, gently mix and prepare to a final volume of 80 μ The reaction solution of L.
Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.
3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.
4) Add 350 to the adsorption column μ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
10. Add 500 to the adsorption column μ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
11. Repeat step 10.
12.
Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.
Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
13. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column μ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.
Attention:
1) The volume of RNase Free Water should not be less than 30 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 30-50 μ Repeat step 13 for the new RNase Free Water.
3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 13.
ZJ25F0825349