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 Digestion Buffer A with Proteinase K was used. Increase the incubation time.
• 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 Columns/wells” 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 or Buffer RL was not added to the lysate.
  Ensure that the appropriate amount of ethanol and Buffer RL are 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.

Clogged columns/wells can result from one or a combination of the following factors:

• Insufficient solubilization of cells or tissues.
  Ensure that the appropriate amount of Digestion Buffer A 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.
• Clarified lysate was not used for the binding step.
  Ensure that after the lysis step the sample is centrifuged if a significant amount of debris is present, and that only the clarified lysate is used in subsequent steps.
• 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:

• FFPE sample is old.
  The quality of RNA purified may drastically decrease in old samples. For best performance, freshly prepared samples are highly recommended.
• 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.
• Prolonged incubation at 80°C.
  In order to reverse formalin cross-links, an incubation at 80°C is required. Do not exceed 15 minutes of incubation at 80°C as this will increase RNA fragmentation.
• 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 Buffer RL.

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 the Well Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
• Formalin crosslink was not completely reversed.
  Ensure sufficient incubation at 80°C is performed in Step 2b in the protocol. Do not exceed 15 minutes of incubation at 80°C as this will increase RNA fragmentation.