Incomplete lysis of cells or tissue - Ensure that the appropriate amount of Lysis Solution 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 also “Clogged Column” below.
An alternative elution solution was used - It is recommended that the Elution Solution 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 - Ensure that 50 mL of 95% ethanol is added to the supplied Wash Solution 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.
If the input is cells growing in a monolyer the 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.
If the input is yeast Lyticase was not added to the Resuspension Buffer - Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer.
If the input is bacteria or yeast - All traces of media was not removed. Ensure that all media is removed prior to the addition of the lysis solution through aspiration.
Why has my column become clogged?
Insufficient solubilization of cells or tissue - Ensure that the appropriate amount of lysis buffer 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 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.
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.
RNA Maxi Column Assembly was tightly capped – Ensure that the cap is loosely placed onto the column assembly so as not to impeded liquid flow.
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 the 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.
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 RNAase content, it is recommended that β-mercaptoethanol be added to the Lysis Solution. f. Lysozyme or lyticase used may not be RNAse-free - Ensure that the lysozyme and lyticase being used with this kit is RNase-free, in order to prevent possible problems with RNA degradation.
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.
When I resolve the RNA on a Bioanalyzer, I am observing some “fuzzy” black background. What can I do to prevent this? This could be due to some ethanol carry over from the isolation, therefore ensure that the dry spin is being performed. If needed, double the time of dry spin from 5 to 10 minutes.