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Old 09-23-2016, 07:00 AM   #1
plyguy69
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Location: america

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Unhappy Primescript Reverse Transcriptase Not Working

We are working on single-cell RNA seq using a version of Simone Picelli modified SMARTer method. Instead of Superscript II we were using Clontech's Primerscript. Unfortunately, the latest batches/ lots have been giving huge nonspecific products around 200-300bp. We switched from Superscript II because they had a problem with bacterial RNA contamination. Is anyone out there using alternate RT enzymes for the single-cell RNA seq protocols? Any response or advise would be greatly appreciated!

I'll try attaching our data below....

Thanks,
ply
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File Type: pdf 2016.8.19.columns.3.4.pdf (53.6 KB, 24 views)
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Old 09-23-2016, 11:44 PM   #2
nucacidhunter
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I think the problem is other modifications not the RT enzyme. Your reactions has produced the large transcript drived cDNA. You can get rid of smaller fragments by bead clean up.

Edit: RT enzyme contamination and alternatives has been discussed:

http://seqanswers.com/forums/showthr...seq#post196705

Last edited by nucacidhunter; 09-24-2016 at 12:20 AM.
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Old 09-26-2016, 07:18 AM   #3
plyguy69
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Default Thanks Nucacidhunter

Those cDNA products shown were analyzed after one SPRI bead clean up. You're right I can remove the majority of the lower bp nonspecific products by a second bead clean up (have tried and it worked). We are just concerned about these products showing up in the new lots of Primescript and don't want to rush into HiSeq runs until we know for sure what's going on....
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Old 09-26-2016, 02:05 PM   #4
nucacidhunter
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Looking at your negative control trace might give some idea about their origin.
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