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Norgen Biotek Corp. is dedicated to providing our customers with first class sample preparation kits for RNA, microRNA, DNA and protein purification, clean-up and concentration and to providing dedicated and expert support services to our customers and partners worldwide.
As a testament of our commitment to quality, Norgen is proud to be an ISO 9001:2008 and ISO 13485:2003 registered company.
It is a pleasure to serve you and we look forward to working closely with you to provide the best sample preparation products for your specific needs. |
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Please contact us at:
info@norgenbiotek.com
Norgen Biotek Corp.
3430 Schmon Parkway,
Thorold
Ontario, Canada, L2V 4Y6
Tel: (905) 227-8848
Fax: (905) 227-1061 |
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- Why am I experiencing poor RNA recovery?
- Incomplete lysis of cells - Ensure that the appropriate amount of Lysis Solution was added to the exfoliated cell pellet.
- Column has become clogged - Do not exceed the recommended amounts of 50 mL of urine or 1 x 106 cells. 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 Buffer 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 20 mL of 95% ethanol is added to the supplied Wash Solution prior to use.
- Low cell density in the sample - The cell number in different urine samples vary. While individuals with various diseases have >1000 exfoliated cells per mL of urine, a healthy male may have a number much lower than the 1000 cells per mL limit. It is possible that the total RNA isolated is not visible when resolved on an agarose gel or detected by spectrophotometry. In such cases, a larger input volume may be used. Alternatively, a more sensitive method such as BioAnalyzer or RT-PCR may be used for detection.
- Why has my column become clogged?
- Insufficient solubilization of cells - Ensure that the appropriate amount of Lysis Solution was added to the exfoliated cell pellet.
- Maximum number of cells exceeds kit specifications - Do not exceed the recommended amounts of 50 mL of urine or 1 x 106 cells.
- 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 20°C may cause precipitates to form that can cause the columns to clog.
- 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.
- The cells are old - Older samples contain prematurely lysed cells which release RNase and can degrade RNA. Fresh urine samples are recommended.
- Why does my RNA not perform well in downstream applications?
- RNA was not washed twice with the provided Wash Solution - Traces of salt from the binding step may remain in the sample if the column is not washed twice 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 used. Perform RNAse-free DNaseI digestion on the RNA sample after elution to remove genomic DNA contamination (Appendix A). It is recommended thatNorgen’s RNase-Free DNase I Kit (Product # 25710) be used for this step.
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