Total RNA Column-Based Extraction Kit

Product Description
This kit enables rapid extraction of high‑quality total RNA from cells, bacteria, and certain animal tissues, without the use of toxic reagents such as phenol or chloroform. The lysis buffer included in the kit contains a potent denaturant and an RNase inhibitor, which efficiently lyse samples and inactivate RNases, thereby preserving RNA integrity throughout the procedure. The isolated total RNA is suitable for a variety of downstream applications, including RT‑PCR, qPCR, Northern blotting, and cDNA library construction. The entire extraction process takes only about 10 minutes, offering simplicity, speed, and excellent reproducibility. Please note that this kit is intended for specific tissue types—namely liver, intestine, brain, spleen, and soft tumor tissues—and is not suitable for tougher tissues such as lung, heart, skin, bone, or muscle.
Order Information

Product ingredients

◆Wash Buffer Before use, add 48 mL of anhydrous ethanol and shake well.

Transportation and Storage
Store at room temperature. Shelf life: 12 months.
Instructions for Use
I. Sample Pyrolysis
1. Adherent cultured cells
(1) Remove the culture medium and wash once with PBS.
(2) Add 500 μL of Lysis Buffer to the culture plate (for samples containing fewer than 3 × 10^6 cells), let it sit horizontally for a short time, then pipette up and down or gently stir several times until the cells are completely lysed. Transfer the lysate to a 1.5 mL centrifuge tube and vigorously pipette up and down repeatedly until the cell clumps are fully dispersed; proceed to Step 2. Note: (1) For cell samples, we recommend culturing cells in a 6-well plate or a 35-mm dish, harvesting them at an appropriate density for RNA extraction. Cells cultured in a 24-well plate can also be used once they reach a confluence of 90% or higher. (2) If direct lysis in the culture vessel is inconvenient, you may use a cell scraper to detach the cells, or…
After trypsin digestion, collect the cells into a centrifuge tube. (3) For small cells such as T cells and B cells, which have very low RNA content, it is recommended to increase the cell number to at least 1 × 10^6 or more.
2. Cells in suspension culture
(1) Transfer 1–3 × 10^6 cells in suspension into a 1.5 mL centrifuge tube, and centrifuge at 1000 g for 1 minute to pellet the cells.
(2) Discard the supernatant, add 500 μL of lysis buffer, pipette up and down vigorously 10 times, vortex for 10 seconds, ensuring complete cell lysis and the absence of visible cell clumps.
Then proceed to Step Two.
3. Bacterial cell
Centrifuge at 5,000 rpm for 3 min to collect an appropriate amount of bacterial cells (＜5×10^8); discard the supernatant, add 100 μL of TE buffer containing lysozyme, pipette up and down to mix thoroughly, and incubate at room temperature for several minutes. Add 500 μL of lysis buffer, vortex vigorously for 20 s, centrifuge at 12,000 rpm for 1–2 min, and transfer the supernatant; then…
Then proceed to Step Two.
4. Animal tissues (suitable for intestine, liver, brain, spleen, and tumors; not suitable for hard tissues such as lung, heart, or skin)
(1) Tissue homogenization: Weigh 10–50 mg of fresh tissue and transfer it into a 1.5 mL centrifuge tube. Add 500 μL of lysis buffer, then thoroughly homogenize the tissue using a tissue homogenizer until no visible tissue fragments remain. Centrifuge at 12,000 rpm for 1–2 minutes.
(2) Liquid nitrogen grinding: Thoroughly grind 10–50 mg of tissue in liquid nitrogen, transfer the resulting powder to a centrifuge tube, add 500 μL of lysis buffer, vortex vigorously for 20 seconds, and centrifuge at 12,000 rpm for 1–2 minutes.
(3) Transfer no more than 400 μL of the supernatant into a new centrifuge tube. Do not pipette up the pellet at the bottom of the tube or any foam.
II. RNA Extraction
1. Cell samples
To more accurately estimate the volume of the cell lysate, add an equal volume of anhydrous ethanol to the lysate, vigorously invert the centrifuge tube several times, or pipette up and down forcefully several times to ensure thorough mixing. Then transfer the mixture to the spin column and proceed to Step 3. [Note]: Precipitation may occur; this is normal and does not affect the extraction process—continue with the subsequent steps.
2. Tissue samples
To more accurately estimate the volume of the lysate, add an equal volume of anhydrous ethanol (or a volume of 70% ethanol) to the lysate, vigorously invert the centrifuge tube several times, or pipette up and down forcefully several times to ensure thorough mixing, then transfer the mixture to the spin column.
3. Centrifuge at 7,000 rpm for 1 min (if liquid remains on the spin column, increase the speed to 12,000 rpm), then discard the supernatant.
4. Add 500 μL of Wash Buffer to the RNA column and centrifuge at 12,000 rpm for 1 minute.
5. Discard the waste liquid, place the RNA column in the collection tube, and centrifuge at 12,000 rpm for 1 minute to completely remove any residual Wash Buffer.
6. Remove the RNA binding column and place it in an RNase-free 1.5 mL centrifuge tube; leave the cap open to air‑dry for 1 minute. Add 25–50 µL of elution buffer to the center of the membrane on the RNA column, and let it stand at room temperature for 1 minute.
7. Centrifuge at 12,000 rpm for 1 min. The sample can be stored long-term at −80°C. [Note]: The volume of elution buffer added should not be less than 25 µL; otherwise, the recovery yield may be affected. To obtain RNA with a higher concentration, the centrifuged RNA solution can be re‑added to the adsorption column and the elution step repeated once.
Precautions
1. This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other such purposes.
2. After adding the Lysis Buffer, be sure to vortex thoroughly to ensure effective RNA extraction. If the mixture is very viscous and difficult to transfer, indicating an excessive sample volume, increase the amount of Lysis Buffer or reduce the sample volume.
3. For cell or tissue lysates, add anhydrous ethanol prior to loading onto the column; after thorough mixing, centrifuge the sample using the spin column.
4. Avoid repeated freeze–thaw cycles of the samples to prevent adverse effects on RNA yield and quality.
5. On RNA Purity : The OD260/OD280 and OD260/OD230 ratios are indicators of RNA purity. For high-quality RNA, the OD260/OD280 ratio typically falls between 1.9 and 2.2, while the OD260/OD230 ratio is greater than 1.8 (with a value exceeding 2.0 indicating particularly pure RNA). Using this kit to extract RNA, measurements obtained with a micro-volume spectrophotometer such as the Nanodrop show an OD260/OD280 ratio between 1.90 and 2.20, and an OD260/OD230 ratio between 2.0 and 2.2, both of which are considered acceptable.
Normal.

6.  On Trace Residues of DNA : During total RNA extraction, it is generally impossible to completely eliminate trace amounts of genomic DNA. With this kit, thanks to its unique buffer system and highly specific binding membrane, the resulting total RNA contains very low levels of DNA contamination, which has minimal impact on most qPCR amplification reactions. If your experiment is particularly sensitive to residual genomic DNA, we recommend the following measures: ① DNase treatment: Use DNase I to digest genomic DNA in subsequent steps. ② Optimize primer design: Select primers that span introns, or design primer pairs with distinct amplicon sizes for genomic DNA and cDNA, thereby preventing DNA from serving as a template in the amplification reaction.

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This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.