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 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.

• Laser-Captured Microdissection: Sample was not incubated at 42°C for 30 minutes

  Ensure that the incubation at 42°C for the removal and lysis of cells from the thermoplastic film.

Column clogging can result from the 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.

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 Column Wash procedure is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

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.

• Improper storage of the purified RNA.

  For short term storage, RNA samples may be stored at –20℃ for a few days. It is recommended that samples be stored at –70℃ for longer term storage.

• Frozen tissues or cell pellets 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.

• 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.

Genomic DNA contamination could occur if 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.

Every 100 µL of Buffer RL can only accept up to 30 µL of more liquid.

• For sorted cells with high volume, it is recommended to pellet them gently, resuspend in an appropriate smaller volume, and then proceed with the correct volume of Buffer RL.
• 350 µL of Buffer RL can be added to a 100 µL sample.

Yes, add buffer RL, vortex to lyse sample and then store at -80°C.

Yes, the kit provides enough extra volume of the elution solution to do this. We recommend checking the concentration of each elution separately before deciding to combine them.