The need for DNAse treatment depends on several factors, like:

• The RNA extraction: some procedures are more effective than others in removing gDNA (e.g., acidic phenol / chloroform extraction). It is therefore recommended to carefully evaluate the optimal RNA extraction method available for the specific sample type used as input.

• The library prep technology: the specific library prep can also influence the impact of gDNA levels on the sequencing results.
  3’ mRNA-Seq library preps (e.g., QuantSeq) are less affected by the presence of low levels of gDNA contamination than random primed whole transcriptome library preps.
  Nevertheless, high gDNA levels will also affect 3’ mRNA-Seq library preps and reduce the quality of the downstream data.

• The sample type: some samples can have a higher DNA / RNA content and therefore be prone to gDNA carry-over during RNA extraction.

When RNA samples contain high levels of gDNA, a DNAse treatment is recommended.

DNAse treatment is always recommended for:

• Samples with high DNA / RNA content, like blood samples.

• Low quality / FFPE samples

• Sample types containing large quantities of short, extra-chromosomal DNA

• Sample undergoing mechanical disruption (due to fragmentation and increased carry-over of the gDNA )

What is the effect of gDNA contamination on my data?

The presence of gDNA contamination can strongly impact the sequencing data and lead to biases and quantification issues in the data analysis.
This will also likely translate into the detection of a high proportion of intronic and intergenic reads and loss of strand specificity.

For more information on gDNA contamination and DNAse treatment, please, check also our RNA LEXICON Chapter #5.