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Old 11-29-2015, 01:11 PM   #171
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Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 208

Originally Posted by kobeho24 View Post
Hi Simone,
I don't know if you noticed that the newly published TGIRT paper in the journal RNA. They use the TGIRT to do the RT of human plasma RNA. And I realized that there is a fragmentation step prior to RT when dealing with whole cell total RNA as they suggested. Thus, on my perspective, defeat its better processivity compared to retroviral RT. You mentioned that the average size of cDNA generated by TGIRT is 4-5k. I just wonder if you have an in-house protocol to do so. It would be much appreciated if you let me know.
BTW, I noticed that the TGIRT can template switch not only RNA but also DNA, which means it might not be compatible with single-cell RNA-seq, unless we extract the total RNA from a single cell and do rRNA-depletion before RT, am I right?

Hi Gary,
yes, I saw the paper, thanks! I didnīt know that the TGIRT can do strand switch on DNA as well (this was not mentioned when I contacted the Lambowitz lab). When I first did my tests some time ago I used tot RNA. Unfortunately, it didnīt work at single cell level (pg) and I just dropped it. therefore, I never sequenced anything and never tried on a real cell. I do have a protocol that worked with 100 ng (!) RNA but after the publication of this new article, I want to try again using some modifications and see if I can get it to work with less RNA. So I canīt really share it right now, sorry!
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