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Old 08-25-2010, 05:28 AM   #1
yuzhang
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Location: Bethesda MD

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Default low 260/230 ratio for RNA-seq sample

I have some RNA samples for RNA-seq library prep. The first problem is I have is some of the RNA have low 260/230 ratio (about 0.2-0.5). Can I go ahead with the standard illumina RNA-seq protocol? The first step is the magnetic polyT beads selection, so the low 260/230 ratio should not matter?

The second question is that I just have about 500ng-1ug total human RNA. Is that a risk for standard illumina protocol since they request for 1ug minimal? Or I have to consider amplification?

Thanks for your reply.
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Old 06-14-2011, 07:15 AM   #2
rabiecqa
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HELLO,
do yo had a solution?
thanks
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Old 06-29-2011, 09:30 AM   #3
whw
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I would go ahead and use the standard protocol. We've made several mRNA libraries with less than the recommended 1ug and they turned out fine. Also, pcr can introduce bias into your samples and you don't want that. Were you thinking about using random primers?

I'm not sure about your 260/230 ratio but the nanodrop gives very rough information in my experience. I would go ahead and try to make your library unless you're seriously concerned about sample purity. You can always verify results using the bioanalyzer before loading the flow cell.
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Old 09-27-2012, 12:20 PM   #4
Katherine.Waugh
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Hello,

Did you go ahead and build libraries with the samples that had low 260/230 ratios? I am having similar issues isolating quality RNA for gene expression profiling.

I isolate total RNA from samples stored in Trizol followed by an extra purification step via Qiagen RNeasy Minielute columns (Cat. # 74204). I obtain small amounts (20-240ng) of RNA according to a nanodrop with a RIN number greater than 9 on the bioanalyzer.

However, on the nanodrop I get a peak a 230 (charcterisitc "shoulder" at 225nm), and a bump and 270nm. I have heard that these two peak problems are normal when isolating small amounts of RNA via Trizol and near impossible to remove.

I would like to use the RNA to make libraries for gene expression profiling on the Illumina HiSEQ but do not want to submit low quality RNA. Did you find a way that I can check ahead of time to make sure that whatever the source of 230 contamination will not affect library construction (RT-PCR?).

I would greatly appreciate any ideas on how to further purify my RNA (butanol/ether experiences?), any past experiences as to whether or not such nanodrop readings predicted poor library construction, and other ways to better purify small samples already stored in Trizol.

Thank you for your time and I hope you were able to get good data from your samples!

Katie
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