Cell and Tissue Total RNA Extraction Kit
25-minute rapid extraction, with no chloroform added.
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Instruction manual- Product Description
- Composition
- Precautions
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Transportation and Storage Conditions: RNAZol Buffer should be transported and stored at 2–8°C; other reagents and spin columns may be shipped at room temperature and stored at room temperature (15–25°C). Elution buffer is recommended to be aliquoted into small portions before first use and stored at room temperature (15–25°C). The shelf life is one year.
Product Overview:
This kit is designed for the rapid extraction of high‑quality total RNA from cell and animal tissue samples. The lysis buffer contains guanidinium isothiocyanate and phenol, which efficiently lyse biological samples and inactivate RNases, ensuring the integrity of the extracted RNA. The kit offers simple, fast operation with stable and reliable performance; it does not require chloroform or time‑consuming alcohol precipitation. The entire extraction process takes approximately 25 minutes, yielding RNA of high yield and purity, suitable for applications such as RT‑PCR, RT‑qPCR, Northern blotting, gene expression microarray analysis, and high‑throughput sequencing.
Preparation Items
1. This kit’s lysis buffer contains phenol. All steps prior to RNA elution must be performed inside a fume hood, and appropriate personal protective equipment—such as protective clothing, gloves, eye protection, and a face shield—must be worn. If the solution comes into contact with the eyes, rinse immediately with copious amounts of water and seek medical attention. If it contacts the skin, wash thoroughly with plenty of detergent and water; if irritation persists, seek medical care.
2. Before first use, add 90 mL of anhydrous ethanol to the Wash Buffer and mix thoroughly.
3. For first-time use, it is recommended to aliquot the Elution buffer into small portions in a clean bench.
Operating Steps
1. Sample homogenization procedure
l Animal Tissue: Transfer 10–50 mg of animal tissue into a centrifuge tube, add 0.5 ml of RNAZol Buffer, and immediately homogenize thoroughly using a pestle or a homogenizer. Tissues such as liver, spleen, and kidney are rich in RNA and proteins; it is recommended to keep the sample amount between 10 and 25 mg.
Reference for Organizational Dosage
Organization Type Liver, pancreas, intestines, stomach, spleen, kidneys Heart, lung muscle Fat Reference dosage (mg) 10-25 mg 20-30 20-50 50 l Adherent cells: Remove the culture medium and wash once with 1× PBS. For a six-well plate (diameter 3.5 cm), add 0.5 ml of RNAZol Buffer to each well, and pipette up and down approximately 10 times to lyse the cells and detach them. (For a 10 cm² culture flask, add 2 ml of RNAZol Buffer.)
l Suspension cells: Centrifuge to pellet the cells (<5×10^6 cells), wash once with 1× PBS, and centrifuge again; discard the supernatant. Vortex or pipette up and down to resuspend the cells. Add 0.5 ml of RNAZol Buffer and pipette up and down approximately 10 times to ensure thorough cell lysis.
2. Add 200 µl of Elution Buffer to 0.5 ml of RNAZol Buffer, invert several times to mix, and incubate at room temperature for 5 minutes.
3. Centrifuge at room temperature at 12,000 × g for 10 minutes.
RNA-binding step
4. Transfer 500 µl of the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of isopropanol, invert several times to mix thoroughly, and transfer the mixture into an RNA purification column (with a collection tube). (Note: The upper aqueous phase is approximately 600 μl; it is recommended to pipette 500 μl to avoid aspirating contaminants. When extracting small‑volume samples, to minimize loss, you may transfer the entire supernatant.)
5. At room temperature, centrifuge at 12,000 × g for 1 minute, and discard the supernatant.
Column Cleaning Procedure
6. Add 500 μl of Wash Buffer to the RNA purification column (including the collection tube), centrifuge at 12,000×g at room temperature for 1 minute, and discard the flow-through.
7. Repeat step 6 once.
8. Place the RNA purification column into the collection tube, and centrifuge the empty column at room temperature at 12,000 × g for 1 minute.
RNA elution step
9. In a clean bench, transfer the RNA purification column into a clean, RNase‑free 1.5 mL centrifuge tube; discard the collection tube, and leave the purification column open to air‑dry for 2 minutes.
10. Add 20–30 μl of Elution Buffer to the central region of the RNA purification column membrane, and let it stand at room temperature for 2 minutes.
11. Centrifuge at room temperature at 12,000 × g for 1 minute, discard the purification column, and collect the RNA in a 1.5 ml centrifuge tube; store at −80°C.
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Product Composition:
Product Number Component (100 times) 400-105A RNAZol Buffer 50 ml 400-105B Wash Buffer 30 ml 400-105C Elution buffer 30 ml 400-105D RNA Purification Column (with Collection Tube) 100 sets -
Precautions
1. The extraction process is carried out at room temperature. Use the recommended sample volume to prevent insoluble precipitates from clogging the spin column. After opening the kit, perform each step strictly according to the protocol to avoid RNase contamination, and aliquot the Elution Buffer into small portions for storage.
2. Before first use, add the appropriate volume of anhydrous ethanol to the Wash Buffer and mix thoroughly.
3. To ensure complete RNA dissolution, the volume of the elution buffer should ideally be no less than 20 μl. Repeated elution can increase RNA yield. The elution buffer must be applied directly to the membrane at the center of the RNA purification column; do not apply it to the side walls.
4. To ensure extraction quality, it is recommended to use RNase‑free reagents and consumables throughout the entire process, particularly during the RNA elution step.
Keywords
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