Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/05

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Summary

Today I'll be checking up on the transfected U2OS cells, hopefully harvesting them for RNA sequencing and quantification. I'll also be checking on the SKNSH culture in the 6-well plate and advancing the KAH126/U2OS cell line.

Goals

  • Fluorescent microscopy of transfected U2OS cells
  • Flow cytometry of transfected U2OS cells
  • RNA isolation/purification
  • Advance KAH126/U2OS culture from PS-β to PS-1
  • Passage U2OS cells from PS-4 to PS-5
  • Monitor progress of SKNSH cells

Protocol

flow cytometry of fluorescent mammalian cells
thawing cells
advancing cells
splitting/passaging cells
RNA purification with TRIzol

Materials

  • List materials here, to be gathered before the experiment
  • Consumables such as media, antibiotics, DNA, kits etc.

Notes

Observations, measurements, placeholder info etc. go here

Results/Conclusions

Phase / mCherry composite images of transfected U2OS cultures below.

well 3

well 4

well 5

It appears that none of the cultures contain adherent transfected cells, as all the fluorescence I can observe is localized in cells and debris that are not part of the confluent monolayer. I'm not certain why this is the case - overcrowding of cells? PcTF activating a gene that inhibits adhesion? Toxicity of PcTF-RFP?

Regardless, I'm not optimistic about getting high quality RNA data from these samples, as cells that are not adherent at this stage of the culture are not likely to be viable or biologically active. Future work might entail splitting the samples 1 day after transfection, in case overcrowding is the issue.


After speaking with Dr. Haynes we've decided to halt work on the transiently transfected U2OS cells and will not collect RNA from the cells or measure their fluorescence via flow cytometry. I'll continue to advance the KAH126/U2OS cell line, passage the unmodified U2OS line and look into using nucleofection on the U2OS cell line from another bioengineering lab.