1. Why am I experiencing poor RNA recovery?
    1. Incomplete lysis of cells or tissue - Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue.
    2. Large RNA Removal Column has become clogged - Do not exceed the recommended amounts of starting materials. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See also “Clogged Column” below.
    3. An alternative elution solution was used - It is recommended that the Elution Solution supplied with this kit be used for maximum RNA recovery.
    4. Low RNA content - Different tissues and cells have different RNA contents. Some tissues may not contain small RNA at detectable levels when processing the small sample sizes required for this procedure.
    5. Flowthrough from the first binding step was discarded - The flowthrough from the binding step with the Large RNA Removal Column contains the small RNA molecules, thus ensure that it is not inadvertently discarded.
    6. Ethanol was not added to the flowthrough before binding to the microRNA Enrichment Column - Ensure that the appropriate amount of ethanol was added to the flowthrough from the first binding step before it is applied to the microRNA Enrichment Column. This is imperative in order to capture the small RNA molecules.
    7. Ethanol was not added to the Wash Solution - Ensure that 50 mL of 95% ethanol is added to the supplied Wash Solution prior to use.
    8. If the input is cells growing in a monolyer the cell monolayer was not washed with PBS - Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells.
    9. If the input is bacteria - All traces of media was not removed. Ensure that all media is removed prior to the addition of the lysis solution through aspiration.
  2. Why has my column become clogged?
    1. Insufficient solubilization of cells or tissue - Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue.
    2. Maximum number of cells or amount of tissue exceeds kit specifications - Refer to specifications to determine if amount of starting material falls within kit specifications.
    3. High amounts of genomic DNA present in sample - The lysate may be passed through a 25 gauge needle attached to a syringe 5-10 times in order to shear the genomic DNA prior to loading onto the Large RNA Removal Column.
    4. Centrifuge temperature too low - Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15°C may cause precipitates to form that can cause the columns to clog.
  3. Why is my RNA degraded?
    1. RNase contamination - RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of the user guide.
    2. Procedure not performed quickly enough - In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly. This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized.
    3. Improper storage of the purified RNA - For short term storage RNA samples may be stored at –20°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.
    4. Frozen tissues or pellets were allowed to thaw prior to disruption - Tissue samples should be flash-frozen in liquid nitrogen and transferred immediately to a -70°C freezer for long-term storage. Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised.
  4. Why does my RNA not perform well in downstream applications?
    1. was not washed 3 times with the provided Wash Solution - Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution. Salt may interfere with downstream applications, and thus must be washed from the column.
    2. Ethanol carryover - Ensure that the dry spin under the Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
  5. Why is there genomic DNA contamination in my RNA? How can I prevent this? Large amounts of starting material were used. Perform RNAse-free DNaseI digestion on the RNA sample after elution to remove genomic DNA contamination. It is recommended that Norgen’s RNase-Free DNase I Kit (Product # 25710) be used for this step.
  6. Why are there large RNA species present in my elution?
    1. Improper amount of ethanol added to the lysate before binding to the Large RNA Removal Column - Ensure that the appropriate amount of ethanol was added to the lysate before it is applied to the Large RNA Removal Column. This is imperative in order to capture the large RNA molecules onto the column.
    2. Large amount of starting material used - Repeat purification using less starting material. Alternatively, the isolation procedure can be repeated using the elution as the input. The elution volume should first be adjusted to 300 µL using the provided Lysis Solution. The procedure can then be followed as written in the manual, starting with the addition of ethanol, centrifuging the lysate in order to pellet any debris, and applying the clarified lysate to the Large RNA Removal Column. Repeating the procedure should result in the removal of the large, contaminating RNA species.