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Old 03-20-2017, 09:26 AM   #1
Seq_Sai
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Default low 260/230 ratios of rRNA depleted and purified plant sample

I am planning to construct libraries for RNAseq from ribosomal RNA depleted plant RNA. The total RNA samples (145ng/ul) were treated with RiboZero kits (illumina) and purified. After the RiboZero treatment, i quantified the RNA samples on Nanodrop, and noticed that the concentration of sample decreased to 53.7 (this was expected), however the 260/280 ratios was 1.78 (previously 1.8) and 260/230 ratios was 0.97( previously 1.7). I am concerned that these ratios would affect the downstream processes. Any ideas why these ratios could have changed? or suggestions on how I can improve these ratios? Any help would be appreciated

PS: RNA sample volume is 8 ul and purification method was ethanol/sodium acetate incubation.

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Old 03-20-2017, 03:52 PM   #2
nucacidhunter
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rRNA makes up more than 80% of total RNA prep. If your input volume also was 8 ul then I would suspect that rRNA depletion has been less successful. You should loos at least 80% of input mass.

low 260/230 ratio could be due to residual sodium acetate from precipitation step or ethanol from wash. This can be corrected by re-precipitation and clean up more carefully but you will loos some RNA. Effect of low 260/230 will depend on the volume of downstream reaction and extent of contaminant.
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