Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/08/18

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Overview

I've made two attempts at transfecting SKNSH cultures with the KAH126-MV2 plasmid this month. I'm presenting the most recent findings now, as they're the most promising, and have some insight as to what's going on with the transfection.

Fluorescence Microscopy

3 days post transfection

Well 1 - KAH126-MV2

Well 2 - KAH126-MV2

Well 3 - no plasmid control

Well 4 - KAH126-MV2

Well 5 - KAH126-MV2

Well 6 - no plasmid control

6 days post transfection

Well 4 - KAH126-MV2

Well 5 - KAH126-MV2

Well 6 - no plasmid control

Flow Cytometry Data

Day 3 post-transfection data: each sample (Wells 1, 2 and 3) was split into 2 fractions. The non-adherent fraction of cells was everything that washed off in the old medium and the PBS rinse. These cells were spun down and resuspended in 1 mL fresh PBS. The adherent fraction was the remaining cells that stuck to the well and were subsequently trypsinized, washed, and resuspended in PBS before analysis on flow cytometry. The adherent and non-adherent fractions have interesting comparisons in terms of fluorescence.

Sample ID Adherent fraction (cells/mL) Non-adherent fraction (cells/mL) Total cell density (cells/mL) Adherent %RFP Non-adherent %RFP Total %RFP
Well 1 3.74E+05 2.39E+05 6.13E+05 4.64 50.10 22.37
Well 2 3.15E+05 2.78E+05 5.93E+05 5.85 54.95 28.87
Well 3 1.04E+06 2.30E+04 1.06E+06 0.01 4.60 0.11

As measured by red fluorescence, the true transfection efficiency appears to be approximately 25% for KAH126-MV2 in SKNSH, but the observed transfection efficiency in terms of cells actually collected for RT-PCR and RNA-seq is much lower, around 5%. Either the PcTF-RFP gene is causing cell death, or the change in gene expression is causing the cells to no longer adhere to their substrate. Either way there's a lot of missing cells that aren't being measured downstream.

If the non-adherent cells are dead, they won't have much high-quality mRNA in them and so processing them doesn't make much sense. If they're alive, however, they will still have good mRNA and so pooling them into the adherent fraction will improve the results.



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