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Old 01-10-2012, 02:43 AM   #1
wzombie
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Default Strange Bioanalyzer results

Hi,

we have some plant RNA samples obtained from a phenol-chloroform extraction followed by LiCl precipitation; we would like to sequence these RNA samples with Illumina Hiseq platform, however RIN ratios are all between 5.5 and 6.5 (Iíve attached a typical bioanalyzer result, Nanodrop values for this sample are 2.14 for 260/280 ratio and 2.41 for 260/230 ratio)

Does anyone know what kind of contamination we have in our samples and to what kind of problems this could lead if we tried to sequence this sample? purification with phenol-choloform and ethanol precipitation does not lead to better results, any idea if there's another possibility to purify the samples?

Thanks a lot for comments - suggestions!
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Old 01-10-2012, 07:38 AM   #2
pmiguel
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Did you do a DNAse treatment of your RNA preps? DNA might be causing your elevated baseline.

About the extraneous peaks, what tissue and types of plants are these?

You will often see mitochondrial and chloroplast ribosomal RNA peaks in plant RNA. That might be what you are seeing. Also, some organisms have different cleavage variants of rRNA. This is common in insects and, I think, some bacteria.

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Old 01-10-2012, 08:14 AM   #3
kmcarr
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Did you use the plant specific rRNA assay for the 2100? Plant chloroplasts have their own set of rRNAs which may be highly abundant in certain tissues. See this AppNote from Agilent about their plant RNA integrity assay.
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Old 01-10-2012, 08:52 AM   #4
wzombie
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Phillip, yes we did a DNAse treatment, then we checked by PCR for DNA contamination with several microsatellites and with an 18S rDNA marker, no fragment was amplified..
These RNA samples were extracted from peach leaves, so yes there might be a lot of mitochondrial and chloroplast rRNA in there, but could they explain the peak at 49 seconds or the low RIN ratios?

kmcarr, yes we also used the plant specific rRNA assay for the 2100, which concluded to very similar electropherograms and very similar RIN values...
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Old 01-10-2012, 09:01 AM   #5
pmiguel
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Past a certain point it is hard to tell. There may just be a highly abundant non-rRNA RNA at 49 seconds. If you know anyone else who has isolated RNA from this tissue, you would want to ask them.

You sample was denatured prior to running, right?

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Old 01-11-2012, 04:24 AM   #6
wzombie
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thanks for your very fast comments!!

well, a few weeks ago i thought that denaturation was responsible for bad results so i also loaded samples without denaturation, but no difference was observed between denaturated and not-denaturated samples..
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