RNA quality (bioanalyser) for agilent microarray

rabiecqa · 04-06-2011, 02:43 AM

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.

pmiguel · 04-06-2011, 05:19 AM

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

rabiecqa · 04-06-2011, 06:27 AM

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 · 04-06-2011, 07:07 AM

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

TonyBrooks · 04-06-2011, 07:18 AM

How about possible DNA contamination? Have you DNase treated your samples?

rabiecqa · 04-06-2011, 07:30 AM

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)

TonyBrooks · 04-06-2011, 07:53 AM

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.

rabiecqa · 04-06-2011, 09:08 AM

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 · 04-08-2011, 02:14 AM

hi, it's was a DNA contamination.. The use of DNAse I solve the problem and there is no strange peak..
thanks for all!

pmiguel · 04-08-2011, 04:51 AM

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

Zapp · 04-15-2011, 04:24 AM

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

Zapp

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04-06-2011, 02:43 AM	#1
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	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.

04-06-2011, 07:07 AM	#4
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	some of additional results there: 1:NK cells 2: PBMCs cells Last edited by rabiecqa; 04-06-2011 at 07:10 AM.

04-06-2011, 05:19 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	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

04-06-2011, 06:27 AM	#3
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	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.

04-06-2011, 07:18 AM	#5
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	How about possible DNA contamination? Have you DNase treated your samples?

04-06-2011, 07:30 AM	#6
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	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)

04-06-2011, 07:53 AM	#7
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	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.

04-06-2011, 09:08 AM	#8
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	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

04-08-2011, 02:14 AM	#9
rabiecqa Junior Member Location: france-Marseille Luminy Join Date: Feb 2011 Posts: 8	hi, it's was a DNA contamination.. The use of DNAse I solve the problem and there is no strange peak.. thanks for all!

04-08-2011, 04:51 AM	#10
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	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

04-15-2011, 04:24 AM	#11
Zapp Junior Member Location: Saudi Arabia Join Date: Mar 2011 Posts: 6	I would rather assume that the RNeasy columns selectively bind low molecular DNA. Zapp