FAQ's for Animal/Cell Total RNA Isolation kit

The following analysis of the problems you may encounter in animal tissue/cell RNA extraction will help you with your experiments. In addition, for other experimental or technical problems in addition to operating instructions and problem analysis, we have dedicated technical support to help you. If you have any needs, please contact us at: 028-83360257 or E-mali : Tech@foregene.com.

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 min after the empty tube centrifugation to maximize the removal of ethanol residue.