Sample quality for RNA_SEQ

cub103 · 07-28-2010, 01:12 PM

Dear all,

I am preparing samples for RNA-SEQ - total RNA was isolated from OCT-Embedded frozen tissue using trizol - I run a RNA integrity check today but there's a lot degradation in some of my samples (See attached, band 1-6). I wonder if these samples could still be used or I have to get some high quality RNA?

BTW, Would too much OCT in the samples affect the isolation process and Quality of RNA?

Thanks.

Cub

ScottC · 07-29-2010, 03:52 PM

Hi,

If you have degraded RNA, then you might want to consider using a method other than poly-A based enrichment (which is what most kits use, including Illumina's sample prep kit) for removing structural RNA and other highly abundant species. There is a method that utilises time- and concentration-dependant rehybridisation and a duplex-specific nuclease to remove highly abundant transcripts. If you're using Illumina sequencing, there is a tech note on it on their website. A google search should turn up the documentation that you need.

Cheers,

Scott.

cub103 · 07-30-2010, 11:13 AM

Hi Scott,

Thanks of the information. I was not able to locate the document you mentioned - wondering if you can post a link? Thank you.

Cub

josdegraaf · 08-02-2010, 07:26 AM

Search for DSN Normalization SamplePrep Application Note

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07-29-2010, 03:52 PM	#2
ScottC Senior Member Location: Monash University, Melbourne, Australia. Join Date: Jan 2008 Posts: 246	Hi, If you have degraded RNA, then you might want to consider using a method other than poly-A based enrichment (which is what most kits use, including Illumina's sample prep kit) for removing structural RNA and other highly abundant species. There is a method that utilises time- and concentration-dependant rehybridisation and a duplex-specific nuclease to remove highly abundant transcripts. If you're using Illumina sequencing, there is a tech note on it on their website. A google search should turn up the documentation that you need. Cheers, Scott.

07-30-2010, 11:13 AM	#3
cub103 Member Location: new orleans, la Join Date: Feb 2010 Posts: 12	Hi Scott, Thanks of the information. I was not able to locate the document you mentioned - wondering if you can post a link? Thank you. Cub

08-02-2010, 07:26 AM	#4
josdegraaf Member Location: Germany Join Date: Mar 2010 Posts: 33	Search for DSN Normalization SamplePrep Application Note