Preparing RNA free of DNA

  • DNA-free RNA preparations are absolutely required for applications like gene expression analysis using PCR-based methods (e.g., real-time PCR).
  • No RNA isolation methods including the most common monophasic lysis reagents-based RNA isolation methods ensure complete removal of DNA from the RNA preparations.
  • Genomic DNA is the major source of DNA contamination in RNA preparations as most RNA preparation methods rely on complete disruption of the cells, leading to the release of genomic DNA from the nuclei. In addition, organelles like mitochondria and chloroplast (in plant cells) can also be the source of DNA. In some special situations (for example cells transfected with plasmid vector/infected with viruses), plasmid DNA/virus DNA can also be the source of contaminated DNA.
  • Therefore, RNA preparations can be subjected to a subsequent purification step that can remove DNA contamination. 
  • There are several methods that can be used to remove DNA from the RNA samples. Some methods are good at removing a bulk amount of DNA whereas others are good at removing trace to moderate amounts of DNA. Often the one good at removing bulk amounts of DNA leave traces of DNA in RNA preparations.
  • Depending on the applications where the RNA preparations to be used, one can choose specific methods.
    • Phenol:Chloroform extraction
    • Lithium chloride (LiCl) precipitation
    • Digestion of RNA samples with DNase I
  • Both Phenol:Chloroform extraction and Lithium chloride (LiCl) precipitation are often used to remove bulk amounts of DNA contamination from the RNA sample. 
  • DNase I treatment is good at removing traces to moderate amounts of DNA from the RNA samples.

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