Why and how are gDNA contaminations in RNAseq library preps bad?

lukas1848 · 11-04-2011, 03:06 AM

Hi,

the title says it all.

I am doing Trizol extractions of total RNA for subsequent RNAseq library preps from small tissue samples. I'm wondering if DNAse treatment and/or LiCl precipitation are necessary to remove residual gDNA from my total RNA extracts.

Once again I'm referring to the Sengupta et al paper (in which they describe the preparation of RNAseq libraries from 10 ng total RNA) as their initial RNA purification is remarkably simple.

protist · 11-04-2011, 04:51 AM

Hi,
Contaminating gDNA can be carried through the RNAseq library generation. For Eukaryotic libraries it can manifest as increased intergenic or intron reads that are not representative of the actual transcriptome. If you are concerned my advice would be check your RNA with a quick RT-PCR.

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11-04-2011, 03:06 AM	#1
lukas1848 Member Location: Germany Join Date: Jun 2011 Posts: 54	Why and how are gDNA contaminations in RNAseq library preps bad? Hi, the title says it all. I am doing Trizol extractions of total RNA for subsequent RNAseq library preps from small tissue samples. I'm wondering if DNAse treatment and/or LiCl precipitation are necessary to remove residual gDNA from my total RNA extracts. Once again I'm referring to the Sengupta et al paper (in which they describe the preparation of RNAseq libraries from 10 ng total RNA) as their initial RNA purification is remarkably simple.

11-04-2011, 04:51 AM	#2
protist Senior Member Location: Ireland Join Date: Jan 2009 Posts: 101	Hi, Contaminating gDNA can be carried through the RNAseq library generation. For Eukaryotic libraries it can manifest as increased intergenic or intron reads that are not representative of the actual transcriptome. If you are concerned my advice would be check your RNA with a quick RT-PCR.