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Old 04-19-2011, 12:11 PM   #1
bunnymac
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Location: Pullman, WA

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Default Dynabeads mRNA isolation problem

I have a problem to isolate mRNA with Dynabeads.

I extracted total RNA from Chlamydomonas with AMBION RiboPure kit.

After that, I purified the total RNAs with Qiagen RNeasy mini kit.

And I used Dynabeads to isolate mRNAs but it showed strange picture.

Its picture is attached.

When I tried to make libraries with these samples, I failed. (No bands)

I checked the total RNAs quality by Bioanalyzer 2100 and they are good.

Does anybody have some idea about this problem?

Thanks in advance,

JJ
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Old 04-19-2011, 05:10 PM   #2
epistatic
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Did you measure your cDNA yield after mRNA capture, frag, and 1st/2nd strand? We like to do that QC step before taking samples through library process. It could also be helpful to run the enriched mRNA on the bioanalyzer. From the picture and failed libraries it would be hard to say what the problem was.
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Old 04-27-2011, 04:54 PM   #3
bunnymac
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Default Bioanalyzer-mRNA

I checked the mRNA level with bioanalyzer.
But I could not get any mRNA with Dynabeads.
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Old 05-09-2011, 10:35 AM   #4
gogreen
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hi bunnymac,
Probably you've figured out the issue already, but I just saw the bioanalyzer profile now and realized that it was a RNA Nano. When you say 4/6/8 ug, do you mean total RNA or mRNA? (I hope it's the first one!) Did you measure the amount of mRNA before running a bioanalyzer?. The RNA Nanos are not quite sensitive for things less than 10 ng which could be the case here (since you do see some faint signal in the last samples). I would try running them on a RNA Pico chip. I use Seramag beads for mRNA purification and with 10 ug of Total RNA, I normally see the mRNA.
Cheers!
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Old 05-09-2011, 10:44 AM   #5
EMONGS
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I agree with go green...and just to add, it is not unusual to not see bands even if the concentration is high. I would start over with more sample; if possible use an invitrogen magnetic rack and just cut the library where you need to after ligation and proceed from there.

All the best!
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Old 05-09-2011, 02:09 PM   #6
gogreen
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hi Emongs, IMHO, the invitrogen magnet doesn't work any better than other ones. I totally quit using invitrogen magnets and now use magnets which costs nothing compared to the terribly expensive invitrogen ones. Even fridge magnets would actually work
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