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Old 03-27-2012, 04:46 PM   #1
libprep
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Default Bioanalyzers: High 28S rRNA Peak

Hi,

I was just wondering if anyone has ever seen a Eukaryote RNA Nano BioAnalyzer with an extremely large 28S peak. The 28S reached 70 fluorescent units and the 18S reached about 15, which resulted in an rRNA ratio for 28s/18s of 8.0. No that is that not the RIN value, that is the value for the rRNA ratio. I have run hundreds of BioAnalyzer chips myself and seen a ton more, but I have never seen ratios this high. Has anyone else ever seen this?

This could just be me being super skeptical, but I don't know if I want to use these samples to make libraries even though everything else about the traces look perfect. Any advice/comments would be greatly appreciated!

Thanks!
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Old 12-17-2012, 04:11 PM   #2
mpappas
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Hi all

I have the very same problem with plant RNA. The 28s/18s ratio is no that high but I have consistently ratios of 3 to 4. Traces looks ok, even leaves samples(BR3-F), with chloroplast rRNA peaks don´t seem to be degraded, RIN values are ok. But I can´t understand the high 28s intensity.

Attached are traces from xylem (BR3-X) and leaves (BR3-F).

I hope to have some help form the experts.
Attached Files
File Type: ppt bioanalyzer-BR3.ppt (623.5 KB, 40 views)
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Old 12-18-2012, 03:35 AM   #3
pmiguel
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I would speculated that in some cases the nuclear (25S) rRNA is co-migrating with the large chloroplast (23S) rRNA. However, one would expect to see either both 18S (nuclear) and 16S (chloroplast) or a combination peak there as well. In some cases you do. But in other cases you don't.
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Old 12-18-2012, 03:49 AM   #4
mpappas
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Thanks for your answer, Philip

Do you have any experience with degradome sequencing? I´m afraid to use this RNA samples. I also tried to use Qiagen columns at the end of my CTAB/BME extraction but I got low 260/230 ratio and I´m afraid to lose important RNAs for degradome sequencing due to size exclusion. What do you think about it?

PS: Send the traces for samples extracted with qiagen columns.
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File Type: ppt bioanalyzer-BR3-qiagen.ppt (440.0 KB, 10 views)
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Old 12-19-2012, 03:32 AM   #5
pmiguel
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Quote:
Originally Posted by mpappas View Post
Thanks for your answer, Philip

Do you have any experience with degradome sequencing? I´m afraid to use this RNA samples. I also tried to use Qiagen columns at the end of my CTAB/BME extraction but I got low 260/230 ratio and I´m afraid to lose important RNAs for degradome sequencing due to size exclusion. What do you think about it?

PS: Send the traces for samples extracted with qiagen columns.
Why do you think that a low 260/230 ratio is an issue? Please be specific -- what contaminating solute that absorbs at 230 nm will interfere with downstream steps?

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Old 12-19-2012, 11:58 AM   #6
mpappas
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I´m just trying to attend all the requirements the company that will prepare the degradome library and sequence it is asking. But, as I mentioned, I´m afraid of using the cloumn and exclude important cleaved RNA for degradome data. That´s why I prefer to use CTAB protocol with isopropanol precipitation. In this case, baseline seemed just a little higher but I think that all the DNA present can higly contribute to that.

Do you understand my point? Is it worth using the columns if I seem to loose lots of information and besides that I have a worse 260/230 ratio? I´m betting I shouldn´t use it.



Marília
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Old 12-20-2012, 03:30 AM   #7
pmiguel
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Quote:
Originally Posted by mpappas View Post
I´m just trying to attend all the requirements the company that will prepare the degradome library and sequence it is asking. But, as I mentioned, I´m afraid of using the cloumn and exclude important cleaved RNA for degradome data. That´s why I prefer to use CTAB protocol with isopropanol precipitation. In this case, baseline seemed just a little higher but I think that all the DNA present can higly contribute to that.

Do you understand my point? Is it worth using the columns if I seem to loose lots of information and besides that I have a worse 260/230 ratio? I´m betting I shouldn´t use it.



Marília
You mean that you want sequence from very short RNA fragments in your sample? If so, most library construction protocols are not going to discard the shorter fragments anyway, using Ampure low-size cut-off purifications after various steps.

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