Effective genomic DNA removal – crucial for accurate gene expression analysis

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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

Genomic DNA removal

Total RNA extraction: effective tissue genomic DNA removal. Total RNA was purified in duplicate from various mouse tissues (10 mg per sample) using the RNeasy Plus Mini Kit or kits from other suppliers. Real-time PCR assays for c-jun were performed to determine the amount of DNA contamination in the purified RNA.

   •  gDNA Eliminator Solution provided in RNeasy Plus Universal Kits

•  cDNA synthesis with integrated gDNA removal using:
   •  Quantitect Reverse Transcription Kit
   •  QuantiNova Reverse Transcription Kit

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.

 

RNA purification
For additional information on gDNA removal, please watch our video or find out more on our dedicated RNeasy Plus family page.

Kjell Kirschbaum

Kjell Kirschbaum, M.Sc., Global Market Manager in the Discovery Sciences, trained as a bioveterinary scientist at the University of Utrecht in the Netherlands. He has hands-on experience in nucleic acid and protein purification, cell culture, PCR and qPCR technology. Kjell joined QIAGEN in 2011 as a telemarketing specialist, regularly interacting with customers about their day-to-day experimental needs and offering relevant solutions. Currently, he is involved in managing global projects for sample preparation and assay technologies.

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