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Old 04-02-2017, 11:20 PM   #245
RevTK
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Location: Basel

Join Date: Apr 2017
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Dear all,
I`m working with laser-capture microdissected cells (e.g. isolated a few hundred cell equivalents from frozen tissue sections) from mouse tissue. I`m using the single-cell lysis buffer from clontech to generate a lysate of my captured cells right after LCM and then use that lysate directly for the cDNA generation. According to this publication, SMART-Seq2 from this lysate should provide decent results: https://www.ncbi.nlm.nih.gov/pubmed/27387371

After 21 preamplification cycles I get the cDNA profiles seen in the attached .pdf.
The kit used is the SMART-Seq2 Ultra-low v3.

Instead of the expected peak at 1.5-2kb, I get a wide range of smaller peaks across the whole range.
Does this mean degraded RNA? Or possibly fragmented due to the UV laser?
Do these profiles look like too many preamp-cycles were used?
In the above mentioned publication, they also seem to get somewhat of a shift towards smaller cDNA fragments when directly lysing 50 to 120 cells.
But they still got good sequencing results.

If my RNA is fragmented, oligo-dT priming / the used kit won`t work properly I assume.

Is there any chance for a successful sequencing from these kinds of samples? Would switching to a totalRNA-kit that uses random priming improve my chances?

Thank you for your help.
Attached Files
File Type: pdf 21preamp_samples.pdf (211.5 KB, 71 views)

Last edited by RevTK; 04-02-2017 at 11:22 PM.
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