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

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RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Main 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_E004mCherryUPL-41, mChF/mChR
Rxn 2:SKNSH_E004GAPDGAPD, Roche GAPD F/R
Rxn 3:SKNSH_C004mCherryUPL-41, mChF/mChR
Rxn 4:SKNSH_C004GAPDGAPD, Roche GAPD F/R
Rxn 5:U2OS_E003mCherryUPL-41, mChF/mChR
Rxn 6:U2OS_E003GAPDGAPD, Roche GAPD F/R
Rxn 7:U2OS_C003mCherryUPL-41, mChF/mChR
Rxn 8:U2OS_C003GAPDGAPD, Roche GAPD F/R
Rxn 9:U2OSKAH126_E001_AmCherryUPL-41, mChF/mChR
Rxn 10:U2OSKAH126_E001_AGAPDGAPD, Roche GAPD F/R
Rxn 11:U2OSKAH126_E001_BmCherryUPL-41, mChF/mChR
Rxn 12:U2OSKAH126_E001_BGAPDGAPD, Roche GAPD F/R
Rxn 13:U2OSKAH126_C001mCherryUPL-41, mChF/mChR
Rxn 14:U2OSKAH126_C001GAPDGAPD, Roche GAPD F/R
1 2 3 4 5 6 7 8 9 10 11 12
ARxn 1Rxn 1Rxn 1Rxn 2Rxn 2Rxn 2Rxn 3Rxn 3Rxn 3Rxn 4Rxn 4Rxn 4
BRxn 5Rxn 5Rxn 5Rxn 6Rxn 6Rxn 6Rxn 7Rxn 7Rxn 7Rxn 8Rxn 8Rxn 8
CRxn 9Rxn 9Rxn 9Rxn 10Rxn 10Rxn 10Rxn 11Rxn 11Rxn 11Rxn 12Rxn 12Rxn 12
DRxn 13Rxn 13Rxn 13Rxn 14Rxn 14Rxn 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_C004mCherryGAPD28.7328.080.63315.70E-021
A1/A4SKNSH_E004mCherryGAPD19.5027.99360.325.97569.160.87
U2OS_C003mCherryGAPD28.6226.310.20151.37E-021
B1/B4U2OS_E003mCherryGAPD19.9227.26161.118.34799.9101.3
U2OSKAH126_C001mCherryGAPD23.0926.5210.741.2861
C1/C4U2OSKAH126_E001_AmCherryGAPD17.1725.87414.52438.615.051
C7/C10U2OSKAH126_E001_BmCherryGAPD17.8226.23340.342.431.75.361



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