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Old 03-07-2014, 02:47 PM   #1
MLog
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Default ribosomal RNA depletion

Hello,
I'm using Epicentre Ribo-Zero kit for removal of rRNA from Drosophila. I checked RNA on Agilent, before and after depletion, and I observed a picture that seems strange for me (attached). A lot of small RNAs that do not seem to be present in the initial sample and nothing that looks like mRNA peak (it should be about 1000-2000 nt, I guess).
What is your opinion, is it normal?
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File Type: jpg Drosophila_depleted.jpg (35.3 KB, 82 views)
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Old 03-13-2014, 02:08 PM   #2
Olaf Blue
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Hi,

Drosophila is not the best substrate for Ribo-Zero, but it does work (see attached data). Not all rRNA species are readily removed, but if you do a Google Scholar search under "Ribo-Zero" and "Drosophila" there are at least one or two papers that cite this combination.

Here's an example:

Kumar et al. BMC Research Notes 2012, 5:230

I hope this is helpful.

Olaf
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File Type: pdf rRNA rmoval test in Drosophila.pdf (45.8 KB, 51 views)
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Old 06-10-2014, 08:14 PM   #3
weigrc
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Hello MLog,

How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


Thanks for the sharing,
Wei
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Old 06-10-2014, 11:04 PM   #4
MLog
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Quote:
Originally Posted by weigrc View Post
Hello MLog,

How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


Thanks for the sharing,
Wei
Hi Wei,
not as good as I wish, actually - about 30-40%. Next time I did double depletion and it worked much better, with ~ 7% mapped ro rRNA.
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Old 06-11-2014, 12:15 AM   #5
weigrc
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Hello MLog,

For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

What is your input amount of the total RNA?

Thanks,
Wei
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Old 06-11-2014, 03:37 PM   #6
MLog
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Quote:
Originally Posted by weigrc View Post
Hello MLog,

For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

What is your input amount of the total RNA?

Thanks,
Wei
I usually take 1-3 micrograms of total RNA. With human and bacterial RNA it worked well - less than 1% of reads mapped to rRNA. Probably for Drosophila it's worth trying to take less.
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