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Old 06-01-2013, 01:03 PM   #1
mdubs
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Default miRNA precipitation prior to Illumina TruSeq Small RNA lib prep

Hi, I have isolated small RNA using the miRvana kit and would like to concentrate my product for input into the TrueSeq Small RNA library prep kit and minimize loss (no use of columns). My question is, has anyone used glycogen (with NaOAC, EtOH) in a precipitation just prior to the library step? My concern is that glycogen may interfere with the the adaptor ligations to come.

I spoke with Illumina and they did not have a clear answer to this. However, I did find an earlier version of their TruSeq Small RNA manual that included glycogen in a concentration step just prior to ligation processes. Although, in the current manual, there is no mention of it. Any info would be much appreciated.

Thanks in advance.
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Old 06-06-2013, 03:52 AM   #2
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Default glycogen or linear acrylamide

You can certainly use glycogen prior to the library step. In my experience, glycogen does not interfere with ligation. You may want to also try out linear acrylamide, its inert and has less of a chance of being contaminated with host nucleic acid.


Quote:
Originally Posted by mdubs View Post
Hi, I have isolated small RNA using the miRvana kit and would like to concentrate my product for input into the TrueSeq Small RNA library prep kit and minimize loss (no use of columns). My question is, has anyone used glycogen (with NaOAC, EtOH) in a precipitation just prior to the library step? My concern is that glycogen may interfere with the the adaptor ligations to come.

I spoke with Illumina and they did not have a clear answer to this. However, I did find an earlier version of their TruSeq Small RNA manual that included glycogen in a concentration step just prior to ligation processes. Although, in the current manual, there is no mention of it. Any info would be much appreciated.

Thanks in advance.
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Old 06-06-2013, 08:17 AM   #3
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We anyone uses the Illumina Small RNA kit is beyond me. It's been published for two years that the ligation bias using that kit gives ENTIRELY WRONG results.

http://www.silencejournal.com/content/3/1/4
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Old 06-07-2013, 08:11 PM   #4
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Here is the original paper that identified the bias:

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

To solve the problem they used adapters with randomized ends.
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Old 06-19-2013, 12:34 AM   #5
peterawe
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Hi. Thanks for the link! The ligation bias is unfortunate and difference in read counts between different miRNAs are not readily interpreted as a measure of their biological expression. However, the profiles generated from these protocols could still be used to access e.g. relative expression between groups.

A question: In the Sorefan et al. Silence Journal paper they used the older Illumina kit, not the current Truseq small RNA. Did Illumina adress some of these issues with the new version?
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Old 06-19-2013, 05:51 AM   #6
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No, unfortunately the ligation bias has not been solved with Illumina's latest TruSeq Small RNA Kit. Relative comparisons between groups can be made with the same microRNAs, but different microRNAs cannot accurately be compared to each other.
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Old 06-19-2013, 06:00 AM   #7
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There are a couple small companies releasing kits with the modified adapters which supposedly reduce or eliminate the ligation bias. We are testing them now and comparing the results to QPCR and microarray expression profiling.
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Old 06-19-2013, 07:24 AM   #8
peterawe
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Thanks for the answer. On the positive side, the Truseq small RNA kit gives in our hand excellent technical reproducibility during library prep - which is also a very important point.

Do you have link to any of the kits you are testing and when do you think your results would be out? I am really interested if you have any experience/data/papers on which kits would perform better. We are about to start profiling a large series and are investigating different options.
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