| Product Description | The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of total RNA. This product combines phenol/guanidine lysis technology and silicon matrix membrane purification technology. The unique lysis solution can effectively inhibit RNases while removing most of DNA and proteins from cell or tissue samples through organic extraction. For some sensitive downstream experiments, if miRNA enrichment is required, this kit can be used to enrich miRNA separately. This product is suitable for a wide range of samples, with high purity of prepared RNA, and can be directly used for sensitive downstream applications, such as Northern Blot analysis, Real Time PCR, Microarray Analysis, etc.
| M665531 | Component | 50 T | Storage | | M665531A | TRIzon Reagent | 60 mL | 2-8℃. Protect from ligt. | | M665531B | Buffer RWT (concentrate) | 15 mL | RT | | M665531C | Buffer RW2 (concentrate) | 11 mL | RT | | M665531D | RNase-Free Water | 10 mL | RT | | M665531E | Spin Columns RM with Collection Tubes | 50 sets | RT | | M665531F | Spin Columns RS with Collection Tubes | 50 sets | RT | | M665531G | RNase-Free Centrifuge Tubes (1.5 mL) | 50 EA | RT |
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Self prepared reagents: chloroform, anhydrous ethanol (newly opened or dedicated for RNA extraction). 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 0.5 M 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 extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of miRNA extraction.
Before the first use, anhydrous ethanol should be added to Buffer RWT and Buffer RW2 according to the instructions on the reagent bottle label.
4. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly. Operation steps:
Protocol A: miRNA enrichment (can be directly used for sensitive downstream experiments)
1. Sample processing
1a Organization: Grind the organization in liquid nitrogen. Add 1 ml of TRIzon Reagent to every 30-50 mg of tissue, shake and mix well. The sample volume shall not exceed one tenth of the volume of TRIzon Reagent.
1b Single layer culture of cells: Remove the culture medium, add TRIzon Reagent, and add 1 ml of TRIzon Reagent every 10 cm2 (the amount of lysis solution depends on the area of the culture bottle). 1c Cell suspension: Centrifuge to obtain cell precipitate, discard supernatant. Add 1 ml of TRIzon Reagent to every 5 x 106-1 x 107 cells (cells do not require washing).
1d Plasma or serum: Take 200 μ Add 5 times the volume of TRIzon Reagent to plasma or serum samples, shake and mix well for 30 seconds.
2. After adding TRIzon Reagent to the sample, blow it repeatedly several times to fully crack it. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Optional steps: Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 5 minutes, take the supernatant, and transfer it to a new centrifuge tube (provided by oneself) (if the sample contains more proteins, fats, polysaccharides, etc., this step can be performed).
4. Add chloroform to the supernatant and add 200 to every 1 ml of TRIzon Reagent used μ Chloroform, cover the tube, vigorously shake for 15 seconds, and let it sit at room temperature for 5 minutes.
Centrifuge at 5.4 ℃ and 12000 rpm for 15 minutes. The sample is divided into three layers: red organic phase, middle layer, and colorless aqueous phase. Transfer the upper colorless aqueous phase to a new centrifuge tube (self prepared).
6. Add 1/3 volume of anhydrous ethanol to the solution obtained in step 5, mix well, and transfer the obtained solution and precipitate together into the adsorption column RM (Spin Columns RM) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the adsorption column RM after centrifugation, and retain the effluent.
7. Add 2/3 times the volume of anhydrous ethanol to the solution obtained in step 6 and mix well.
8. Transfer the solution and precipitate obtained from the previous step into the adsorption column RS (Spin Columns RS) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
9. Add 700 to the adsorption column RS μ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
10. Add 500 to the adsorption column RS μ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.
11. Repeat step 10.
12. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RS 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 RS, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.). 13. Place the adsorption column RS 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 the obtained RNA solution 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 RS and repeat step 13
Protocol B: Extraction of total RNA (including miRNA and other small molecule RNAs<200 nt), steps 1-5 are the same as protocol A.
6. Add 1.25 times the volume of anhydrous ethanol to the solution obtained in step 5 and mix well.
7. Transfer the solution and precipitate obtained from the previous step into the spin columns RM that have been loaded into the collection tube. If you cannot add all the solution to the adsorption column RM at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
8. Add 700 to the adsorption column RM μ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
9. Add 500 to the adsorption column RM μ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.
10. Repeat step 9.
11. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RM at room temperature for a few minutes to thoroughly air dry.
Attention:
The purpose of this step is to remove residual ethanol from the adsorption column RM, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
12. Transfer the adsorption column RM into 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 the obtained RNA solution 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 12 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 RM and repeat step 12.
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