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

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qPCR Control Plate for mCherry & GAPD targets Main 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

Image: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
TRUEA1/A4U2OS_E003 1:10mCherryGAPD20.037299120.112905421.054
TRUEA7/A10U2OS_E003 1:100mCherryGAPD22.8665175323.184345751.246
TRUEB1/B4U2OS_E003 1:1000mCherryGAPD26.6381953227.282233811.563
TRUEB7/B10U2OS_C003 1:10mCherryGAPD29.2048010319.513571421.21E-03
TRUEC1/C4U2OS_C003 1:100mCherryGAPD28.9431357222.166218679.12E-03
TRUEC7/C10U2OS_C003 1:1000mCherryGAPD29.0959423723.688829362.36E-02
TRUED1/D4U2OSKAH126_E001_A 1:10mCherryGAPD17.6245829918.988101172.573
TRUED7/D10U2OSKAH126_E001_A 1:100mCherryGAPD21.6421356922.820955752.264
TRUEE1/E4U2OSKAH126_E001_A 1:1000mCherryGAPD24.2921481425.912153973.074
TRUEE7/E10U2OSKAH126_E001_B 1:10mCherryGAPD18.5198561319.70827542.279
TRUEF1/F4U2OSKAH126_E001_B 1:100mCherryGAPD20.6196638421.88602162.406
TRUEF7/F10U2OSKAH126_E001_B 1:1000mCherryGAPD24.8242695626.353744262.887
TRUEG1/G4U2OSKAH126_C001 1:10mCherryGAPD23.5165969719.45462055.99E-02
TRUEG7/G10U2OSKAH126_C001 1:100mCherryGAPD26.1954645522.477072617.60E-02
TRUEH1/H4U2OSKAH126_C001 1:1000mCherryGAPD28.4033204626.659315890.2985


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