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.
• Column has become clogged. Either the Genomic DNA Removal Column or the RNA Purification Column may become clogged if the recommended amount of starting materials is exceeded. 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.
• Ethanol was not added to the lysate. Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
• 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.
• Low RNA content in cells or tissues used. Different tissues and cells have different RNA contents, and thus the expected yield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material.
• 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.
• Yeast: Lyticase was not added to the Resuspension Buffer. Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer.
• Bacteria and Yeast: All traces of media not removed. Ensure that all media is removed prior to the addition of 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 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 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 were allowed to thaw prior to RNA isolation. Do not allow frozen tissues to thaw prior to grinding with a mortar and pestle in order to ensure that the integrity of the RNA is not compromised.
• Starting material may have a high RNase content. For starting materials with high RNase content, it is recommended that β-mercaptoethanol be added to the Lysis Solution.
• Lysozyme or lyticase used may not be RNase-free. Ensure that the lysozyme and lyticase being used with this kit are RNase-free, in order to prevent possible problems with RNA degradation.

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 plate is not washed 3 times with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.
• 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.

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.

The Total RNA Purification "Plus" kit comes with gDNA removal columns for efficient removal of genomic DNA.