SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq: Comparing Next-Generation Sequencing and Microarray Technologies in a Toxico Newsbot! Literature Watch 0 08-13-2011 04:10 AM
RNA-Seq: ExpressionPlot: A web-based framework for analysis of RNA-Seq and microarray Newsbot! Literature Watch 0 07-30-2011 04:00 AM
Directly compare microarray expression and RNA-Seq data? kidderb Bioinformatics 0 06-30-2011 08:52 AM
PubMed: Power of deep sequencing and agilent microarray for gene expression profiling Newsbot! Literature Watch 0 05-09-2010 08:00 PM

Reply
 
Thread Tools
Old 04-06-2011, 02:43 AM   #1
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default RNA quality (bioanalyser) for agilent microarray

hi,
i have this profile of RNA quality to preceed for microarray , one strange peak is apearing every time.
can you help me?
the picture is there.
rabiecqa is offline   Reply With Quote
Old 04-06-2011, 05:19 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

There are several possibilities. It probably is not a result of RNA degradation because you have clean large and small sub-unit rRNA peaks that appear in roughly the expected stoichiometry.

Possibly the sample has been ribo-depleted prior to the Bioanalyzer run? A single cycle of most ribo-depletion methodologies will leave some rRNA behind. The result could look like the image you show above. Then the answer would be "that is your non-ribosomal RNA".

Alternatively there may be some other RNA that is being transcribed to extremely high levels in your samples. These could include some highly induced gene, viral RNA, contamination from previous RNA preps done in the same centrifuge tube.

Finally, you do not say what species this RNA is from. The large sub unit rRNA looks a little smaller than I would expect for a mammal. If this RNA is of mammalian origin, the sample may have been eukaryotic-rRNA depleted (eg, Invitrogen ribo-minus) but still retain some mitochondrial rRNA peaks. Or, alternatively, there may be some bacterial component to the total RNA. Again, a ribo-depletion method that was eukaryotic rRNA-specific would remove the eukaryotic rRNA, but perhaps not the bacterial rRNA.

If you gave us a little more information about the sample (is it total RNA or has it been ribo-depleted? what species and tissue is it from? Etc.) the possibilities would be narrowed.

--
Phillip
pmiguel is offline   Reply With Quote
Old 04-06-2011, 06:27 AM   #3
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default

hi,
The samples were freezed in 10% DMSO and Fetal bovine serum .Then the total RNA was extracted from human Peripheral Blood Mononuclear cells(PBMC) by using "RNaseasy Qiagen mini Kit.The problem is majority of the samples have this additional peak.Please give me some suggesions.
rabiecqa is offline   Reply With Quote
Old 04-06-2011, 07:07 AM   #4
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default

some of additional results there:
1:NK cells
2: PBMCs cells

Last edited by rabiecqa; 04-06-2011 at 07:10 AM.
rabiecqa is offline   Reply With Quote
Old 04-06-2011, 07:18 AM   #5
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

How about possible DNA contamination? Have you DNase treated your samples?
TonyBrooks is offline   Reply With Quote
Old 04-06-2011, 07:30 AM   #6
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default

i have not treated with DNAse. do you think i should treat it ?
because the experiment was in sterile environment (PSM: post security microbiology Level 2)
rabiecqa is offline   Reply With Quote
Old 04-06-2011, 07:53 AM   #7
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

I would. You could always treat a few samples with DNase I, stop the reaction with EDTA, then re-run on the Bioanalyser to check if there's an improvement.
TonyBrooks is offline   Reply With Quote
Old 04-06-2011, 09:08 AM   #8
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default

Yes I'll treat with DNAse I because there some informations contort this idea and seem to be the same problem of mine.
link of information: http://www.ambion.com/techlib/tn/112/10.html
rabiecqa is offline   Reply With Quote
Old 04-08-2011, 02:14 AM   #9
rabiecqa
Junior Member
 
Location: france-Marseille Luminy

Join Date: Feb 2011
Posts: 8
Default

hi, it's was a DNA contamination.. The use of DNAse I solve the problem and there is no strange peak..
thanks for all!
rabiecqa is offline   Reply With Quote
Old 04-08-2011, 04:51 AM   #10
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Good call Tony!
Mysterious to me though. Genomic DNA I expect to be mainly >50kb -- which would likely not even show up on a nanochip. If something degraded the bacterial chromosome down to ~1 kb, I guess that is what you would see. Maybe there was some DNAse in earlier steps, but it did not result in a complete digestion?

--
Phillip
pmiguel is offline   Reply With Quote
Old 04-15-2011, 04:24 AM   #11
Zapp
Junior Member
 
Location: Saudi Arabia

Join Date: Mar 2011
Posts: 6
Default

I would rather assume that the RNeasy columns selectively bind low molecular DNA.

Zapp
Zapp is offline   Reply With Quote
Reply

Tags
bioanalyser rna quality

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 07:28 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO