Poor RNA recovery could be due to one or more of the following:

• Incomplete lysis of cells or tissue. Ensure that the appropriate amount of Buffer RL was used for the amount of cells or tissue.
• 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 FAQ related to “Clogged Column” below.
• An alternative elution solution was used. It is recommended that the Elution Solution A supplied with this kit be used for maximum RNA recovery.
• 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.
• 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.
• 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.
• Ethanol was not added to the Wash Solution A. Ensure that 90 mL of 96 – 100% ethanol is added to the supplied Wash Solution A prior to use.
• Cell Culture: 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.
• Bacteria: All traces of media not removed. Ensure that all media is removed prior to the addition of the Buffer RL through aspiration.

Column clogging can result from one or a combination of the following factors:

• Insufficient solubilization of cells or tissues. Ensure that the appropriate amount of Lysis Buffer was used for the amount of cells or tissue.
• Maximum number of cells or amount of tissue exceeds kit specifications. Refer to specifications to determine if the amount of starting material falls within kit specifications.
• 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.
• Centrifuge temperature is 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.

RNA can be degraded due to the following factors:

• 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 this user guide.
• 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.
• 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.
• 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.

If the RNA does not perform well in downstream applications, it may be due to one or more of the following:

• RNA was not washed 3 times with the provided Wash Solution A. Traces of salt from the binding step may remain in the sample if the well is not washed 3 times with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the well.
• Ethanol carryover. Ensure that the dry spin under Column Wash in the centrifugation protocol or the extended vacuum in the vacuum protocol is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

The contamination with genomic DNA may be due to large amount of starting material used. Perform RNase-free DNase I 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.

Large RNA species may be present in the elution due to:

• 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.
• 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 Buffer RL. 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.

Although it is recommended to use fresh non-coagulating (EDTA) blood for RNA purification, frozen blood samples can also be processed. Please thaw the frozen blood samples on ice for 15 minutes, and then at room temperature (21 degrees) for 10 minutes before proceeding to the kit protocol. Please note that frozen blood samples are known to yield more degraded RNA compared to fresh blood samples.