Whether you are working with formalin-fixed, paraffin embedded (FFPE) tissue or normal tissue samples, the elimination of genomic DNA (gDNA) from your RNA preps is crucial to obtain reliable results in gene expression analysis, especially if you want to determine the gene expression pattern in a specific cell or under specific conditions.
Possible consequences of not removing gDNA:
• Copurification with RNA and overestimation of RNA yield
• Overestimation of transcript abundance in qRT-PCR
• Higher background in your microarray or NGS experiment, leading to false results
And even worse…not being able to publish your results in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. These guidelines ensure the integrity of the scientific literature and increase experimental transparency.
Luckily, there are several ways to eliminate gDNA from your RNA sample. You can perform:
• DNase treatment on-membrane or on-bead
• DNase treatment in-solution, especially for large amounts of DNA
• Chemically separate DNA and RNA
• Organic extraction, e.g., QIAzol Lysis Reagent
• gDNA Eliminator spin columns provided in RNeasy Plus Micro or Mini Kit
• gDNA Eliminator Solution provided in RNeasy Plus Universal Kits
Another way to avoid gDNA amplification is to carefully design your qPCR primers so they span an exon-exon junction. By selecting a large intron, the distance between the primers will be large enough to avoid gDNA amplification.