Guides for problems analysis

The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。

No RNA can be extracted or the yield of nucleic acid is low

There are usually many factors that affect recovery efficiency, such as: sample RNA content, method of operation, elution volume, etc.。

Analysis of common causes：

    1.Ice bath or low-temperature (4 ° C) centrifugation during operation. 

Suggestion: Room temperature (15-25 ° C) operation, never ice bath and low temperature centrifuge. 

     2. Improper sample storage or sample storage for too long. 

Suggestion: Store samples at -80 ° C or freeze in liquid nitrogen, and avoid repeated freeze-thaw use; try to use freshly collected samples for RNA extraction.  

     3.Insufficient sample lysis

Recommendation: Please ensure that the sample and the working solution (Linear Acrylamide) have been thoroughly mixed and incubated for 10 min at room temperature (15-25 ° C)

    4.The eluent was added incorrectly 

Recommendation: Make sure that RNase-Free ddH2O is added to the middle of the membrane of the purification column

    5.Improper volume of anhydrous ethanol in Buffer viRW2

Suggestion: Please follow the instructions, add the correct volume of anhydrous  ethanol to Buffer viRW2 and mix them well before using the kit。

    6.Improper sample usage. 

Suggestion: 200µl of sample per 500μl of Buffer viRL. Excessive sample volume will result in reduced RNA extraction rate.

    7.Improper elution volume or incomplete elution. 

Suggestion: The eluent volume of the purification column is 30-50μl; if the elution effect is not satisfactory, it is recommended to add pre-heated RNase-Free ddH₂O and extend the time placing at room temperature, such as 5-10min

    8.Purification column has ethanol residue after rinsing in Buffer viRW2.

Suggestion: If ethanol still remains after rinsing in Buffer viRW2 and empty-tube centrifugation for 2min, the purification column can be left at room temperature for 5min after empty-tube centrifugation to fully remove remaining ethanol.

The degradation of purified RNA molecules

The quality of the purified RNA is related to factors such as sample storage, RNase contamination, and operation. 

Analysis of common causes：

    1.The collected samples were not saved in time.

Suggestion: If the sample is not used in time after collection, please store it at -80 ℃ or liquid nitrogen immediately. For the extraction of RNA molecules, try to use freshly collected samples whenever possible. 

    2.Collected samples were freezing and thawing repeatedly.

Suggestion: Avoid repeated freezing and thawing (no more than once) during sample collection and storage, otherwise the yield of nucleic acid will decrease. 

     3.RNase was introduced in the operating room or no disposable gloves, masks, etc. were worn.

Suggestion: The extraction of RNA molecules experiment is best performed in a separate RNA operation room, and the experimental table is cleaned before the experiment. Wear disposable gloves and masks during the experiment to avoid RNA degradation caused by RNase introduction. 

      4.The reagent is contaminated by RNase during the use.

Suggestion: Replace with new Viral RNA Isolation Kit for related experiments. 

      5.The RNase contamination of the centrifuge tubes, pipette tips, etc. Suggestion: Make sure that the centrifuge tubes, pipette tips, and pipettes are all RNase-Free.

The purified RNA molecules affected downstream experiments

The RNA molecules purified by the purification column will affect downstream experiments if there are too much salt ions or proteins, such as: reverse transcription, Northern Blot, etc.。

      1.There are remaining salt ions in the eluted RNA molecules.

Recommendation: Make sure that the correct volume of anhydrous ethanol has been added to Buffer viRW2, and wash the purification column twice according to the correct centrifugation speed on the operating instructions；If there are still salt ions remaining, you can add Buffer viRW2 to the purification column, and leave it at room temperature for 5min. Then perform centrifugation to remove salt ions contamination to the greatest extent

      2.There are remaining ethanol in the eluted RNA molecules

Suggestion: once confirming that purification columns have been rinsed by Buffer viRW2, perform empty-tube centrifugation according to the centrifugal speed on the operating instructions. If there is still ethanol remaining, it can be left for 5 minutes at room temperature after an empty-tube centrifugation to remove the remaining ethanol to the greatest extent.

Instruction Manuals:

Viral RNA Isolation Kit Instruction Manual