UltraPure Total RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2102-20
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UltraPure Total RNA Extraction Kit(SpinColumn)
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20T
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CNY470
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R2102-50
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UltraPure Total RNA Extraction Kit(SpinColumn)
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50T
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CNY780
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R2102-200
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UltraPure Total RNA Extraction Kit(SpinColumn)
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200T
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CNY2800
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
IProduct Introduction：
The High-Purity Total RNA Rapid Extraction Kit is suitable for the extraction of total RNA from various animal and plant tissues as well as cells. Lysis Buffer RL can efficiently lyse tissue cells and inactivate RNases. Combined with specific spin columns, it enables the extraction of high-purity, high-quality total RNA free from contaminants such as proteins, genomic DNA and cellular metabolites.
The extracted total RNA features good integrity and is free from protein and DNA contamination, making it suitable for various routine molecular biology experiments, including RT-PCR, Real Time PCR, Northern blot, Dot blot, poly A selection, in vitro translation, cDNA library construction, etc.
Product Features：
1.The silica matrix membrane in the spin column is made of high-quality specially-made adsorption membrane, with minimal variation in adsorption capacity between different columns.
2.It combines the advantages of the guanidine isothiocyanate/phenol one-step method (good reagent stability and high purity) and the convenience of spin columns. It eliminates the need for isopropanol precipitation and ethanol washing steps. RNA is directly eluted from the spin column, avoiding the problem of difficult dissolution caused by over-drying.
3.The unique formulation of Lysis Buffer RL can effectively eliminate genomic DNA contamination.
4.Multiple column washing steps remove impurity proteins and cellular metabolites, resulting in high-purity RNA extraction.
It effectively reduces the content of 5S RNA in the total RNA, improving the RNA purity.
Operation Steps：
Pre-experiment Preparation
1.Use sterile RNase-free centrifuge tubes, pipette tips and other disposable plastic consumables as much as possible. If glassware or non-disposable tools are used, they should be pre-treated with DEPC or decontaminated to remove RNases.
(1) Immerse the glassware and tools to be treated in 0.1% DEPC (diethyl pyrocarbonate) aqueous solution and incubate for 12 hours protected from light.
(2) Autoclave the treated items at 121°C for 30 minutes to remove residual DEPC.
2.Wear lab coat, disposable latex gloves and disposable mask during reagent preparation and experimental operation to avoid artificial RNase contamination.
Sample Pre-treatment
1.Tissue Homogenization
a. Plant Tissues: Take fresh plant tissues and grind them thoroughly in liquid nitrogen, or cut the plant tissues into small pieces and grind them rapidly directly in Lysis Buffer RL. Add 1 mL of Lysis Buffer RL per 50-100 mg of tissue and mix well. Note: The sample volume should generally not exceed 10% of the volume of Lysis Buffer RL.
b. Animal Tissues: Take fresh or -70°C frozen animal tissues and cut them into small pieces as much as possible. Add 1 mL of Lysis Buffer RL per 30-100 mg of tissue and homogenize with a homogenizer. Alternatively, grind the tissues in liquid nitrogen and add 1 mL of Lysis Buffer RL to mix well. Note: The sample volume should generally not exceed 10% of the volume of Lysis Buffer RL.
c. Monolayer Cultured Cells (Adherent Cells): Remove residual culture medium as thoroughly as possible. Add 1 mL of Lysis Buffer RL directly to a 3.5 cm diameter culture plate to cover the cells, and pipette repeatedly to lyse the cells. The volume of Lysis Buffer RL required is determined by the area of the culture plate rather than the number of cells (1 mL per 10 cm²). Insufficient Lysis Buffer RL may lead to DNA contamination in the extracted RNA.
Note: Adherent cultured cells often cannot be completely detached from the culture flask (dish), which does not indicate incomplete lysis. At this time, the cell membranes have actually been completely broken and RNA has been released, so the experiment can be continued.
d. Suspension Cells: Collect cells by centrifugation. Lyse the cells by pipetting repeatedly in Lysis Buffer RL. Add 1 mL of Lysis Buffer RL per 5~10×10⁶ animal cells, plant or yeast cells, or per 1×10⁷ bacterial cells. Avoid washing the cells before adding Lysis Buffer RL, as this will increase the possibility of mRNA degradation. Homogenizer may be required to lyse some yeast and bacterial cells.
e. For bacterial RNA extraction, the Bacterial RNA Extraction Kit (Spin Column Type) (Cat. No. R2120) is recommended. For blood RNA extraction, the TRI Whole Blood (Liquid Sample) Total RNA Extraction Kit (Cat. No. R2108) is recommended.
Extraction Steps
2.Vortex the homogenized sample vigorously and incubate at room temperature for 5 minutes to completely dissociate ribosomes.
3.Optional Step: Centrifuge the sample at 12,000 rpm for 10 minutes at 4°C and collect the supernatant. Centrifugation is recommended if the sample contains high levels of proteins, fats, polysaccharides, or materials such as muscle and plant tubers/nodules. The pellet after centrifugation contains cell membranes, polysaccharides and high-molecular-weight DNA, while the supernatant contains RNA. For adipose tissue samples, remove the upper layer of oil and take the clear homogenate for the next step.
4.Add 0.2 mL of chloroform per 1 mL of Lysis Buffer RL. Cap the tube tightly, vortex vigorously for 15 seconds and incubate at room temperature for 3 minutes.
5.Centrifuge the mixture at 12,000 rpm for 10-15 minutes at 4°C in a high-speed refrigerated centrifuge. After centrifugation, the mixture separates into three layers: the lower red organic phase, the middle phase and the upper colorless aqueous phase. RNA is present in the upper aqueous phase. The volume of the aqueous phase is approximately 50-60% of the volume of Lysis Buffer RL added.
6.Carefully transfer the aqueous phase to a new RNase-free centrifuge tube (do not touch the middle phase). Record the volume of the aqueous phase, add anhydrous ethanol equal to 0.5 times the volume of the aqueous phase, and mix immediately by pipetting.
7.Transfer the mixture (less than 700 μL per time, can be added in two batches) to the Spin Column SpinRA. Centrifuge at 12,000 rpm for 45 seconds, discard the waste liquid and place the spin column back into the collection tube.
8.Add 500 μL of Deproteinization Buffer RE, centrifuge at 12,000 rpm for 45 seconds at room temperature and discard the waste liquid.
9.Add 500 μL of Wash Buffer RW (please check whether anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 45 seconds and discard the waste liquid.
10.Repeat Step 9 once.
11.Place the Spin Column SpinRA back into the empty collection tube, centrifuge at 13,000 rpm for 2 minutes at room temperature to remove as much wash buffer as possible, preventing residual ethanol in the wash buffer from inhibiting downstream reactions.
12.Take out the Spin Column SpinRA (avoid touching the waste liquid in the collection tube with the bottom of the spin column) and place it into a new RNase-free centrifuge tube. Add 50~80 μL of RNase-free H₂O dropwise to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute. After obtaining the RNA, store the RNA solution at -80°C to prevent degradation.
The larger the elution volume, the higher the elution efficiency. If higher RNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30 μL. Excessively small volume will reduce RNA elution efficiency and yield.
