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Old 10-07-2019, 03:33 PM   #2
cmbetts
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Location: Bay Area

Join Date: Jun 2012
Posts: 113
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1. Sure, you can swap in different length RT primers without a noticeable impact on performance
2. SSII is definitely better than SSIII, but I don't know about IV
3. I personally like Takara's SeqAmp, but I used to work there and am biased

A followup question. Why are you adding the cost/complexity of preamplification when gene specific RT-PCR works at the single cell level? Banking the cDNA or assessing multiple genes per cell?

Also, are you sure you don't need NGS level resolution for when you're describing? Single cell methods are going to have lots of allelic dropouts that have nothing to do with actual biological regulation, just good ol' sampling noise, especially for anything that isn't highly expressed. The cost of the number of cells, TOPO cloning, and Sanger reactions required to get a remotely robust answer is going to approach the cost of an NGS run real fast (especially if you use targeted amplicons).

Regardless of sequencing method, I also highly recommend putting UMIs in your RT primer and targeting the 3'UTR, RACE style, if there distinguishing alleles close enough to the end of the transcript. Just watch out for alternative polyA usage and bad annotation. Otherwise, you may falsely assume you've reached a high enough count to assess statistical significance when you've really just been looking at a much smaller population that won the sampling lotto (and PCR jackpot)

Last edited by cmbetts; 10-07-2019 at 03:38 PM.
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