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Old 10-02-2019, 08:41 AM   #1
kumard
Junior Member
 
Location: Midwest USA

Join Date: Jun 2019
Posts: 6
Default RNA purification, DNA contamination, and small RNA sizing profiles

Hello all,

I am extracting total RNA from tissues derived from wild animals after 24-48 h of death. I am able to get a RIN of approximately 7, but my samples have DNA contamination. I am using only 2.5mg of tissue (homogenized in 1 mL of Trizol-like proprietary reagent).


I have tried Trizol-based method (without DNase treatment) that claims to remove DNA and a silica column based method (with On-column DNase treatment from two different suppliers). My RNA concentration approximately 150 ng/uL and DNA contamination is approximately 3 ng/uL (2% of total RNA). Is this level of contamination "tolerable" for RNASeq (both large and miRNA).

I know that getting rid of DNA completely is not possible. But, what should I do now?

My second issue is about change in the small RNA profiles as seen in this image below. The column-based purification is certainly changing/reducing the small RNA species. I do not want to use the columns for this reason, but DNA contamination is giving me a headache now.


I want to get feedback and guidance from those who have first-hand experience in extracting RNA, performing QC, and generating cDNA libraries for RNASeq.

It seems to me that the companies advertise false information and claims are generally not true (May be true for RNA extraction from model organisms and cells) and one need to develop a custom-made solution for their use.

Could someone please also guide me to the published literature on such problems in RNASeq?

Have a nice day!

Thanks,
DK
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