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.
  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.
• 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 tissue used.
  Different tissues 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.
• Insufficient solubilization of tissues.
  Ensure the lysate is diluted with the appropriate amount of RNase-free water, and that the appropriate amount of Proteinase K is added. Also ensure that the Proteinase K treatment is performed at 55°C for the full 15 minutes. The incubation time can be increased up to 30 minutes if required.

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

• Insufficient solubilization of tissues.
  Ensure the lysate is diluted with the appropriate amount of RNase-free water, and that the appropriate amount of Proteinase K is added. Also ensure that the Proteinase K treatment is performed at 55°C for the full 15 minutes. The incubation time can be increased up to 30 minutes if required.
• Maximum 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. Also, ensure that the on-column DNase treatment is performed if high amounts of genomic DNA are found in the sample.
• 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 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 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 2 times with the provided Wash Solution A.
  Traces of salt from the binding step may remain in the sample if the column is not washed 2 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.

Genomic DNA contamination could be caused by the following:

• Large amounts of starting material used.
  Perform the RNase-free DNase I digestion on the RNA sample suggested in the protocol to remove genomic DNA contamination.
• DNase I mix did not completely pass through the column during DNase I treatment.
  Ensure the entire 115 µL of DNase I mix passes through the column. If needed, spin at 14,000 x g (~14,000 rpm) for 1 minute.