| Product Description | Product content:
| Component |
R665870
50 preps |
| RNApure Circulating Reagent |
50 ml | |
Product Introduction:
The free RNA (serum, plasma, urine) extraction reagent is particularly suitable for isolating and purifying total RNA, including microRNAs and other small RNAs (<200nt), from serum and plasma. This product flexibly handles samples with different starting amounts, effectively preserving the integrity of RNA while effectively cleaving the samples. The extracted total RNA has good integrity and is free of protein and DNA contamination. The extracted RNA can be used for downstream experiments such as RT-PCR, Northern Blot, and molecular cloning.
Self prepared reagents: chloroform, isopropanol, 75% ethanol, water without RNase (newly opened or dedicated for RNA extraction). Matters needing attention:
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 yield and quality of RNA extraction.
3. This product contains phenol, which is toxic and corrosive. If inhaled, in contact with the skin, swallowed, etc., it can cause poisoning, burns, and other bodily injuries. When using this product, protective equipment should be worn, such as protective clothing, gloves, eye masks, masks, etc. If you accidentally come into contact with your eyes, immediately rinse with plenty of water and seek medical treatment.
4. After the sample is homogenized with free RNA (serum, plasma, urine) extraction reagent, if chloroform is not added immediately, it can be left at -70 ℃ for more than a month.
5. RNA precipitates stored in 75% ethanol can be stored for one week at 2-8 ℃ and for one year at -20 ℃. RNA has a relatively short half-life and is prone to degradation. It is recommended to conduct subsequent experiments as soon as possible after extraction, such as reverse transcription into cDNA, Northern Blot, etc.
6.If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase. Operation steps:
1. Take 200 μ Add 3 times the volume of free RNA (serum, plasma, urine) extraction reagent to fresh or frozen serum or plasma. Shake for 30 seconds and mix thoroughly.
Attention: After adding free RNA (serum, plasma, urine) extraction reagents to the sample, precipitation may occur. After shaking and mixing, the precipitation basically disappears. If there is still a small amount of precipitation that does not affect downstream experiments, the operation can continue.
2. Leave the processed sample at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Add chloroform to the above solution, add 0.2 ml of chloroform to every 1 ml of serum/plasma sample specific total RNA extraction reagent, cover the tube cap, vigorously shake for 15 seconds, and leave at room temperature for 2-3 minutes.
Attention: If vortex mixing is not possible, you can manually quickly invert and mix for 2 minutes.
4.4 ℃ and 12000 rpm for 20 minutes. At this time, the sample is divided into three layers: red organic phase, middle layer, and upper layer colorless aqueous phase. RNA is mainly in the aqueous phase, and the aqueous phase is transferred to a new RNase free centrifuge tube (self prepared).
5. Add an equal volume of isopropanol to the obtained aqueous solution, invert and mix well, and let it stand at room temperature for 30 minutes. Or precipitate overnight at -20 ℃ for better results.
6.4 ℃ and 12000 rpm for 20 minutes, discard the supernatant.
Note: RNA precipitation before centrifugation is often invisible, and after centrifugation, gel like precipitates are formed on the side and bottom of the tube.
7. Add 75% ethanol (prepared with water without RNase) to wash the precipitate. Wash the precipitate with 1 ml of 75% ethanol for every 1 ml of free RNA (serum, plasma, urine) extraction reagent used.
8.4 ℃ and 12000 rpm for 3 minutes, be careful to aspirate and discard the supernatant, and be careful not to aspirate and discard RNA precipitates.
Attention: The remaining small amount of liquid can be briefly centrifuged and then sucked out with a gun tip, being careful not to discard the sediment.
9. Leave at room temperature for 2-3 minutes and let it dry. Join 30-100 μ Dissolve RNA thoroughly in water without RNase, and store the resulting RNA at -70 ℃ to prevent degradation.
Attention: Do not excessively dry the sediment to avoid difficulty in dissolution. |
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