Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/04/22

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Owwnotebook icon.pngqPCR Control Plate for mCherry & GAPD targets Report.pngMain project page
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Overview

To establish some quality control standards for the RNA-seq and qPCR experiments, I performed qPCR on all dilutions of all templates using mCherry & GAPD as targets.

Templates to be used are:

  • ✓ U2OS (1:10, 1:100, 1:1000)
  • ✓ KAH126-U2OS (1:10, 1:100, 1:1000)
  • SK-N-SH (1:10, 1:100, 1:1000)

Also will be trying out a duplex reaction with GAPD & mCherry primers in the same sample, using a template that shows strong mCherry signal.

qPCR plate layout for U2OS & KAH126-U2OS templates

PCR Plate layouts Nyer Expt01.png

qPCR results for U2OS & KAH126-U2OS templates

Experiment: DualColorProbe_UPL_DNyer042215 Active filters: FAM (465-510), VIC / HEX / Yellow555 (533-580)

Chart Pairing Sample Name Targets References Mean Cp Mean Cp Target/Ref Status
TRUE A1/A4 U2OS_E003 1:10 mCherry GAPD 20.0372991 20.11290542 1.054
TRUE A7/A10 U2OS_E003 1:100 mCherry GAPD 22.86651753 23.18434575 1.246
TRUE B1/B4 U2OS_E003 1:1000 mCherry GAPD 26.63819532 27.28223381 1.563
TRUE B7/B10 U2OS_C003 1:10 mCherry GAPD 29.20480103 19.51357142 1.21E-03
TRUE C1/C4 U2OS_C003 1:100 mCherry GAPD 28.94313572 22.16621867 9.12E-03
TRUE C7/C10 U2OS_C003 1:1000 mCherry GAPD 29.09594237 23.68882936 2.36E-02
TRUE D1/D4 U2OSKAH126_E001_A 1:10 mCherry GAPD 17.62458299 18.98810117 2.573
TRUE D7/D10 U2OSKAH126_E001_A 1:100 mCherry GAPD 21.64213569 22.82095575 2.264
TRUE E1/E4 U2OSKAH126_E001_A 1:1000 mCherry GAPD 24.29214814 25.91215397 3.074
TRUE E7/E10 U2OSKAH126_E001_B 1:10 mCherry GAPD 18.51985613 19.7082754 2.279
TRUE F1/F4 U2OSKAH126_E001_B 1:100 mCherry GAPD 20.61966384 21.8860216 2.406
TRUE F7/F10 U2OSKAH126_E001_B 1:1000 mCherry GAPD 24.82426956 26.35374426 2.887
TRUE G1/G4 U2OSKAH126_C001 1:10 mCherry GAPD 23.51659697 19.4546205 5.99E-02
TRUE G7/G10 U2OSKAH126_C001 1:100 mCherry GAPD 26.19546455 22.47707261 7.60E-02
TRUE H1/H4 U2OSKAH126_C001 1:1000 mCherry GAPD 28.40332046 26.65931589 0.2985