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Old 02-08-2018, 10:32 AM   #6
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Location: Basel (Switzerland)

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Originally Posted by alekzs View Post
I have tried increasing the PCR cycles up to 23 but still got this double-bump pattern.
The samples were frozen, I sort directly into lysis buffer (max 5min per plate), directly spin the plate down and then freeze on dry-ice and transfer to -80C until processing. For processing, I start out with bringing the RNA beads to room temp, RNAzap the work area, let the plates thaw on ice for 10min and then start the protocol.
I donīt have access to the paper you refer to but vaguely remember it.
I have some comments and suggestions:
- why do you sort in such a large volume of buffer. We generally sort in 2 ul (384w plates) or 4 ul (96w plates).
- use RNAse inhibitor at all stages, even during cell isolation. Is your FACS sorter clean...really clean? Always use negative controls in your test: 1 droplet of FACS buffer in a well with lysis buffer, to check that nothing gets amplified.
- use a dead/live marker. PI, CellTracker, whatever works (no Hoechst. itīs toxic and gives often issues)
- sorry my silly question but does "2.2X SPRI" mean? are you doing a bead purification before RT? I wouldnīt transfer and/or remove the content of the plate until after PCR, once you have enough DNA.
- I hope you have a mistake in the short protocol you posted and are not doing RT at 52 deg with Maxima H- but at 42 instead...
- Itīs also not necessary to do 2 rounds of bead purification after PCR. If you are concerned with primer dimers use biotinylated primers and decrease the amount. But this of course, has no influence on the quality of your RNA of just saves you time.

That said, frozen cells always have a much lower viability than fresh ones (a nice comparison here: PMID:28249587). For T cells we generally do 23-24 PCR cycles and you are doing the same, so no issue here. And yes, the "double bump" is a mix of degraded DNA (RNA) and leftover primers probably not removed after purification. Most likely you have a huge excess of unused primers after PCR.

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