Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/23

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Summary

Today we'll be harvesting the U2OS cells from the first transfection attempt. Fluorescence microscopy of the cells indicates there is little or no PcTF-RFP expression in the line, but we'll be doing a run through with flow cytometry and RNA purification regardless, as practice for future attempts.

Goals

  • Harvest U2OS cells from 1st transfection attempt
  • Flow cytometry on cells to determine gene expression levels
  • RNA purification on cells to create cDNA library (see below, Results section)

Protocol

flow cytometry of mammalian cells for measuring fluorescent gene expression
RNA extraction / purification

Materials

  • PBS buffer
  • Trypsin / EDTA
  • Complete cell medium
  • FACS Buffer - 1% FBS in 1x PBS (stored at 4°C)
  • FACS tubes with strainer caps - 6 mL capacity (EMS 64750-25)
  • TRIzol / RNA purification miniprep & supplies
  • pipette tips, etc.

Notes

Overview of flow cytometry data:



Negative control does not have significantly different fluorescence values than the samples (data not shown).

Results/Conclusions

Since the control has the same fluorescence levels as the samples (and therefore the same gene expression levels of PcTF-mCherry), we will not be performing the RNA extraction / purification today. Instead we'll be repeating this experiment tomorrow on the second set of transfected cells.


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