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

1. Homogenization was incomplete.
   Depending on the sample, further vortexing with the flat bed vortex or bead beater equipment may be required. However, it is not recommended to increase the vortex time to longer than 10 minutes at maximum speed.
2. Column has become clogged.
   Do not exceed the recommended input amount of 200 mg of stool. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels.
3. An alternative Elution Buffer was used.
   It is recommended that the Elution Buffer E supplied with this kit be used for maximum RNA recovery.
4. Ethanol was not added to the lysate.
   Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
5. 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.

RNA can be degraded due to following factors:

1. 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 the product protocol documentation.
2. 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.
3. DNase used may not be RNAse-free.
   Ensure that the DNase being used for the optional On Column DNA Removal step is 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:

1. RNA was not washed with Binding Buffer B and Wash solution A.
   Traces of humic acids and salt from the binding step may remain in the sample if the column is not washed with Binding Buffer B and 2 times with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.
2. 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.

Yes, the Stool Total RNA Purification Kit is compatible with samples preserved with Norgen's stool preservative. Please refer to our Application Note here

Yes, our Stool Total RNA Purification kit has been used successfully with bacterial pellets resuspended and stored in RNAlater®. Please contact our technical support team at support@norgenbiotek.com and ask for reference publications.