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Old 06-02-2014, 08:19 AM   #20
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
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Quote:
Originally Posted by jwfoley View Post
Maybe one possible problem is the LNAs in the TSO? This is prone to strand invasion (doi:10.1186/1471-2164-14-665), so the LNAs are not a good idea if you care about full-length transcripts. That's why all the other template-switching protocols use riboguanines. If the strand invasion is biased toward specific sites, that could explain these problems.

EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.
We use a LNA-containing TSO and always have a better coverage at the 5ī-end of transcripts than the Clontech kit which uses 3 riboG.

In our hands, blocking the TSO led to a (slightly) lower gene detection at all RPKM levels and a (slightly) higher variation between replicates. The unblocked LNA-containing TSO was still performing better, therefore we donīt block the TSO unless the "hedgehog" signal shows up.
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