Cell Total RNA Isolation Kit Total RNA Isolation Purification Kits from cell
This kit uses the spin column and formula developed by Foregene, which can efficiently extract high-purity and high-quality total RNA from cells cultured in 96, 24, 12, and 6-well plates.
The kit provides an efficient DNA-Cleaning Column, which can easily separate the supernatant and cell lysate, bind and remove genomic DNA. The operation is simple and time-saving.
The RNA-only Column can efficiently bind RNA with a unique formula. A large number of samples can be processed simultaneously.
*Please wear gloves and take protective measures during the operation as Buffer cRL1 and Buffer RW1 contain irritating chaotropic salts.
■ The whole process is operated at room temperature (15-25℃), without ice bath and low temperature centrifugation.
■ The whole kit is RNase-Free, no need to worry about RNA degradation.
■ DNA-Cleaning Column specifically binds DNA, so that the kit can remove genomic DNA contamination without adding additional DNase.
■ High RNA yield: RNA-only Column and unique formula can efficiently purify RNA.
■ Fast speed: easy to operate and can be completed in 11 minutes.
■ Safety: No organic reagent is required.
■ High quality: The purified RNA is of high purity, free of protein and other impurities, and can meet various subsequent experiments.
It is suitable for extraction and purification of total RNA from cultured cells in 96, 24, 12, and 6-well plates.
The agarose gel battery diagram of Cell Total RNA Isolation Kit treated the above different numbers of cells, 20μl volume elution, take 2μl purified total RNA 1%.
Storage and shelf life
The kit can be stored for 12 months at room temperature (15–25 ℃) or 2–8 ℃ for longer time(24 months).
Buffer cRL1 can be stored at 4 ℃ for 1 month after adding 2-hydroxy-1-ethanethiol(optional).
RNA is not extracted or RNA yields are low
There are often a variety of factors that affect recovery efficiency, such as: tissue sample RNA content, method of operation, elution volume, etc.
1. Ice bath or cryogenic (4 °C) centrifugation was performed during operation.
Recommendation: Operate at room temperature (15-25 ° C) throughout the whole process, do not ice bath and centrifuge at low temperatures.
2. Improper sample preservation or excessive sample storage time.
Recommendation: Store samples at -80 °C or freeze in liquid nitrogen and avoid repeated freeze-thaw use; try to use fresh tissue or cultured cells for RNA extraction.
3. Insufficient sample lysis.
Recommendation: When homogenizing tissue, ensure that the tissue is sufficiently homogenized and that the tissue cells are sufficiently split to explain the release of RNA.
4. The eluent is not added correctly.
Recommendation: Confirm that RNase-Free ddH2O is added dropwise to the middle of the purification column membrane.
5. The correct volume of absolute ethanol was not added to Buffer RL2 or Buffer RW2.
Recommendation: Follow the instructions, add the correct volume of absolute ethanol to Buffer RL2 and Buffer RW2 and mix well before using the kit.
6. Tissue sample dosage is not appropriate.
Recommendation: Use 10-20 mg of tissue or (1-5) × 106 cells per 500 μl buffer RL1, as excessive tissue use can result in reduced RNA extraction.
7. Improper elution volume or incomplete elution.
Recommendation: The elution volume of the purification column is 50-200 μl; if the elution effect is not satisfactory, it is recommended to extend the room temperature placement time after adding preheated RNase-Free ddH2O, e.g. for 5-10 min.
8.The purification column has ethanol residue after Buffer RW2 wash.
Recommendation: If there is ethanol residue after Buffer RW2 washing, empty tube centrifugation for 1min, the time for the empty tube centrifugation operation can be increased to 2min, or the purification column can be placed at room temperature for 5 min to adequately remove the residual ethanol.
Purified RNA is degraded
The quality of the purified RNA is related to factors such as the preservation of the sample, RNase contamination, and manipulation,etc.
1. Tissue samples are not kept in time.
Recommendation: If tissue samples or cells are not used in a timely manner after collection, immediately cryopreserve at -80 °C or liquid nitrogen. To extract RNA, use a newly taken tissue or cell sample whenever possible.
2. Repeated freeze-thawing of tissue samples.
Recommendation: When storing tissue samples, it is best to cut them into small pieces for preservation, and remove one of the pieces when using them to avoid repeated freeze-thawing of the sample and the degradation of RNA.
3. RNase is introduced or not wearing disposable gloves, masks, etc. during the operation.
Recommendation: RNA extraction experiments are best performed in separate RNA manipulation rooms and the table is cleared before the experiment.
Wear disposable gloves and masks during the experiment to minimize RNA degradation caused by the introduction of RNase.
4. Reagents are contaminated with RNase during use.
Recommendation: Replace with a new Animal Total RNA Isolation Kit for related experiments.
5. The centrifuge tubes, tips, etc. used in RNA manipulation are contaminated with RNase.
Recommendation: Confirm that the centrifuge tubes, tips, pipettes, etc. used in RNA extraction are all RNase-Free.
Purified obtained RNA affects downstream experiments
RNA purified by the purification column, if the salt ions, protein content is too large will affect the downstream experiment, such as: reverse transcription,Northern Blot et al.
1. The elutioned RNA has salt ion residues.
Recommendation: Confirm that the correct volume of ethanol has been added to Buffer RW2 and perform 2 purification column washes at the centrifugal speed indicated for operation; if there is any salt ion residue, leave the purification column to Buffer RW2 for 5 min at room temperature and perform centrifugation to maximize the removal of salt contamination.
2. Ethanol residue in elutioned RNA.
Recommendation: Confirm that after buffer RW2 washing, perform the empty tube centrifugation operation at the centrifugation speed indicated for operation, increase the time of the empty tube centrifugation operation to 2 min if there is still ethanol residue, or leave it at room temperature for 5 m