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Gene Expression Assay and Data Analysis
When I follow your recommended protocol of isolating total RNA from mammalian tissues, first using Trizol reagents, then with RNeasy columns, I sometimes see a reduced recovery off the RNeasy columns.
Trizol reagents and RNeasy columns are based on very different principles for nucleic acids purification. RNeasy columns exclude certain contaminants that may give rise to a falsely higher spectrophotometric reading, including carried-over phenol and transcripts shorter than 200 nucleotides in length. These shorter transcripts include the 5S rRNA and tRNA molecules that may account for 10% or more of the total RNA isolated.
To verify that the RNA of interest has been cleaned up efficiently during column purification, it may be helpful to run aliquots of your samples on a gel or perform some gene-specific real-time PCR quantitation. In addition, you can estimate how much total RNA you anticipate to recover since the yield is highly dependent on tissue type. These reference numbers can be obtained through your own experience or can be found in published literature, for example, the RNeasy Mini HandBook.
If you continue to observe significant loss of material on RNeasy columns, please contact QIAGEN Technical Support directly.