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Old 09-21-2016, 11:09 PM   #1
ccrachel
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Default odd Agilent Bioanalyzer results-total eukaryotic(3D culture) RNA pico assay

Hello all! I am a phd student , very new to this field... I was growing some 3D spheroids in a Matrigel droplet and I was testing two samples to prepare them for my institution facility which will prepare library (mRNA) for HisSeq Illumina. My two samples(loaded in lane1 and lane3) was extracted using the mirVana rna extraction kit by ambion, invitrogen, eluted in 95oC nuclease-free water, followed by DNA removal by the invitrogen Turbo kit. On nanodrop measurement, the two samples gave concentration readings of 2.6 and 5.4 ng/uL respectively, with 260/280 being 4.71 and 2.3 respectively. However, when the bioanalyzer(pico assay) result from core facility came back, the profile looks very strange and I couldnt identify the RNA bands due to presence of multiple peaks like noise. Does this mean there is no RNA? or is there presence of some contaminants in my sample that I can further treat before having another go at bioanalyzer analysis? i dont really know...Very Grateful for any advice/help!

thanks a lot in advance

-ry
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Old 09-22-2016, 12:08 AM   #2
nucacidhunter
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Bioanalyzer run has failed and to know if it was sample or assay set up you need to look at ladder run and other samples in the same Chip. If it is your sample try cleaning with a column and rerun.
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Old 09-22-2016, 04:52 AM   #3
microgirl123
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Based on the Nanodrop readings, your RNA concentration is pretty low, and your 260/280 is awful (especially the one that is 4.71). What are your 260/230 ratios, and what do the Nanodrop graphs look like?
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Old 09-22-2016, 01:43 PM   #4
nucacidhunter
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Nanodrop 260/230,280 ratios are invalid when ng/ul value is below 10-15.
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