Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/02/24

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Owwnotebook icon.pngRNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Report.pngMain project page
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Summary

In paragraph format, state what you are going to do today so a reader unfamiliar with your project could understand what is going on.



Goals

  • ✓ Expand K562 cells into T-175 flask
    • Tomorrow (and daily after), measure cell density via FC; when culture density 0.5-1E6 cells/mL, transfer to fresh medium and transfect with lipofectamine
  • ✓ Make 1:10, 1:100, 1:1000 dilutions of cDNA templates
  • ✓ qPCR on all templates with mCherry & GAPD targets; mCherry use 1:10 diluted template, GAPD use 1:1000 diluted template



Protocol

qPCR UPL Assay

expanding cell lines

Note: To expand from T-75 to T-175, I added 20mL of medium to the T-175 and then transferred 5mL of the cell culture to that, for a 1:5 dilution into 25mL total.




Notes

qPCR layout:

Template Gene Target Oligos
Rxn 1: SKNSH_E004 mCherry UPL-41, mChF/mChR
Rxn 2: SKNSH_E004 GAPD GAPD, Roche GAPD F/R
Rxn 3: SKNSH_C004 mCherry UPL-41, mChF/mChR
Rxn 4: SKNSH_C004 GAPD GAPD, Roche GAPD F/R
Rxn 5: U2OS_E003 mCherry UPL-41, mChF/mChR
Rxn 6: U2OS_E003 GAPD GAPD, Roche GAPD F/R
Rxn 7: U2OS_C003 mCherry UPL-41, mChF/mChR
Rxn 8: U2OS_C003 GAPD GAPD, Roche GAPD F/R
Rxn 9: U2OSKAH126_E001_A mCherry UPL-41, mChF/mChR
Rxn 10: U2OSKAH126_E001_A GAPD GAPD, Roche GAPD F/R
Rxn 11: U2OSKAH126_E001_B mCherry UPL-41, mChF/mChR
Rxn 12: U2OSKAH126_E001_B GAPD GAPD, Roche GAPD F/R
Rxn 13: U2OSKAH126_C001 mCherry UPL-41, mChF/mChR
Rxn 14: U2OSKAH126_C001 GAPD GAPD, Roche GAPD F/R
1 2 3 4 5 6 7 8 9 10 11 12
A Rxn 1 Rxn 1 Rxn 1 Rxn 2 Rxn 2 Rxn 2 Rxn 3 Rxn 3 Rxn 3 Rxn 4 Rxn 4 Rxn 4
B Rxn 5 Rxn 5 Rxn 5 Rxn 6 Rxn 6 Rxn 6 Rxn 7 Rxn 7 Rxn 7 Rxn 8 Rxn 8 Rxn 8
C Rxn 9 Rxn 9 Rxn 9 Rxn 10 Rxn 10 Rxn 10 Rxn 11 Rxn 11 Rxn 11 Rxn 12 Rxn 12 Rxn 12
D Rxn 13 Rxn 13 Rxn 13 Rxn 14 Rxn 14 Rxn 14 - - - - - -
E - - - - - - - - - - - -
F - - - - - - - - - - - -
G - - - - - - - - - - - -
H - - - - - - - - - - - -

Template cDNA for reference gene (GAPD) diluted 1:1000
Template cDNA for target gene (mCherry) diluted 1:10

Results/Conclusions

qPCR Results:

Experiment: DualColorProbe_UPL_DNyer022415 Active filters: FAM (465-510), VIC / HEX / Yellow555 (533-580)

Pairing Sample Name Targets References Mean Cp Mean Cp Target/Ref Target/Ref Error Normalized Normalized Error Status
SKNSH_C004 mCherry GAPD 28.73 28.08 0.6331 5.70E-02 1
A1/A4 SKNSH_E004 mCherry GAPD 19.50 27.99 360.3 25.97 569.1 60.87
U2OS_C003 mCherry GAPD 28.62 26.31 0.2015 1.37E-02 1
B1/B4 U2OS_E003 mCherry GAPD 19.92 27.26 161.1 18.34 799.9 101.3
U2OSKAH126_C001 mCherry GAPD 23.09 26.52 10.74 1.286 1
C1/C4 U2OSKAH126_E001_A mCherry GAPD 17.17 25.87 414.5 24 38.61 5.051
C7/C10 U2OSKAH126_E001_B mCherry GAPD 17.82 26.23 340.3 42.4 31.7 5.361