Incomplete lysis of leukocytes - Ensure that the appropriate amount of Binding Solution was used to lyse the leukocyte pellet.
Lysis of red blood cells was incomplete - Ensure that the blood sample is collected with the appropriate amount of EDTA, which will prevent coagulation of the red blood cells and allow for proper lysis. Also check that the appropriate amount of RBC Lysis Buffer is added to the blood sample, and that it is mixed and incubated properly.
Ethanol was not added to the lysate - Ensure that 200 µL of 95-100% ethanol is added to the lysate before binding to the column.
Ethanol was not added to the Wash Solution - Ensure that 50 mL of 95% ethanol is added to the supplied Wash Solution prior to use.
An alternative elution solution was used - It is recommended that the Elution Buffer supplied with this kit be used for maximum RNA recovery.
The column has become clogged - Do not exceed 2 mL of blood or 3 x 106 leukocytes per column. The amount of blood used may need to be decreased if the column shows clogging below the recommended level. See also “Clogged Column” below.
Why has my column become clogged?
lysis of leukocytes - Ensure that the appropriate amount of Binding Solution was used to lyse the leukocyte pellet.
Lysis of red blood cells was incomplete - Ensure that the blood sample is collected with the appropriate amount of EDTA, which will prevent coagulation of the red blood cells and allow for proper lysis. Improperly lysed red blood cells will clog the column.
Amount of blood used exceeds kit specifications - It is recommended that no more than 2 mL of blood or 3 x 106 leukocytes be used in order to prevent possible clogging of the column.
Centrifuge temperature 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.
Why does the cloudy pink solution not become clear red during RBC lysis?
This is caused by incomplete red blood cell lysis. The solution should become a translucent red colour after RBC Lysis Solution has been added and incubated with the blood. If not, pellet the leukocytes and remove as much of the supernatant as possible. Add another 5 volumes of RBC Lysis solution and incubate again.
Why is the leukocyte pellet red?
This is caused by incomplete red blood cell lysis The leukocyte pellet should be white, with only residual traces of red blood cells. If red blood cell lysis is incomplete, the pellet will be red. In this case resuspend the leukocyte pellet in another 5 volumes of RBC Lysis Solution and incubate at room temperature for another 5 minutes.
Why is my RNA degraded?
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°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.
Leukocyte pellets were too old - Leukocyte pellets generated at the end of Step 1 may be stored for up to 2 weeks at -70°C and used in this procedure. It is not recommended that samples be frozen for longer than 2 weeks, as the integrity of the RNA will be compromised.
Why does my RNA not perform well in downstream applications?
RNA was not washed 3 times with the provided Wash Solution - Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution. 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.
Why is there genomic DNA contamination in my RNA? How can I prevent this? Large amounts of starting material were used. Perform RNAse-free DNaseI 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.