Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/26

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Summary

Still checking on the cultures, going to harvest the ones induced to create PcTF-RFP later in the week if their expression levels look good. Also submitting the RNA harvested last week to DNASU, where they'll create cDNA and then quantify it using real-time PCR.



Goals

  • Monitor cultures (SKNSH, U2OS, & KAH126-U2OS)
  • Photograph fluorescence of transfected/induced cultures
  • Harvest cultures that are on day 4 of transfection/induction
  • Quantify RNA prior to submission to DNASU lab for sequencing



Protocol

example protocol




Notes

Observations, measurements, placeholder info etc. go here.



Results/Conclusions

Culture Status

  • SKNSH (6-well plate): 25% confluence, replace with fresh medium
  • SKNSH (T-75): 50% confluence, replace with fresh medium
  • KAH126-U2OS (T-75): 80% confluence, replace with fresh medium
  • U2OS (T-75): 100% confluence, passage

RNA Quantification

Replicate 1 Replicate 2 Measurement
Well 1 - dox 0.95 0.577 260
0.475 0.276 280
2 2.089 260/280
760.119 461.95 ng/µL
Well 2 - dox 0.661 0.647 260
0.313 0.308 280
2.11 2.101 260/280
528.495 517.669 ng/µL
Well 4 - negative control 0.908 0.457 260
0.443 0.224 280
2.048 2.037 260/280
726.307 365.656 ng/µL

"Negative control" is a sample not treated with doxycycline but still containing RNA.

Samples have been submitted to DNASU for creation of cDNA followed by RT-PCR.


Fluorescence microscopy images of KAH126-U2OS cells after 4 days of induction:

Well 1 (negative control) Well 2 (negative control)

Well 3 Well 4

Well 5 Well 6


Fluorescence microscopy images of U2OS cells 4 days after transfection:

Well 1 Well 2

Well 3 Well 4

Well 5 (negative control) Well 6 (negative control)