User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/16

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05/16/14

  • qRT-PCR: QC_plate, all cDNA batches



RT-PCR: QC_plate, all cDNA batches

  • Repeat from 5/14/14, except use 1:100 cDNA dilutions
  • System: Roche LC480 (machine & reagents)
  • Summary: Run mCh, GAPD reactions on all cDNA batches, all cell lines: 16 total. Will be used to compare cDNA quality, PcTF expression, and generate reference values
  • Experiment file name: DualColorProbe_UPL_Haynes051614
  • Target gene wells: mCherry wells: 16x3 = 48; GAPD wells: 16x3 = 48
  • cDNA dilution: 1:100 for all reactions
  • Plate layout: Plate_layouts_Carly.xlsx/QC_plate


cDNA Batches:

  1. K562_E001 (E = experimental, +PcTF)
  2. K562_C001 (C = control, no PcTF)
  3. K562_E002
  4. K562_C002
  5. K562_E003
  6. K562_C003
  7. SKNSH_E001
  8. SKNSH_C001
  9. SKNSH_E002
  10. SKNSH_C002
  11. SKNSH_E003
  12. SKNSH_C003
  13. U2OS_E001
  14. U2OS_C001
  15. U2OS_E002
  16. U2OS_C002


Target Gene Primer/Probe Master mixes (2 tubes)

Reagent (Single well) Target Gene (x48.5) Roche GAPD (x48.5)
2x LC480 Probes Master(7.5 μL)363.75363.75
20 μM Forward primer(0.3 μL)14.5514.55 GAPD primers
20 μM Reverse primer(0.3 μL)14.550
10 μM UPL probe(0.3 μL)14.5514.55 GAPD probe
PCR H2O(0.1 μL)4.8519.40
Total vol.(8.5 μL)412.25412.25

Template Master Mixes (16 tubes)

Reagent (Single well) cDNA Template (x6.5)
diluted Batch# cDNA(2.0 μL)13.0
PCR H2O(4.5 μL)29.3
Total vol.(6.5 μL)42.3


RESULTS

  • Notes: Filters - mCh is FAM (465-510), GAPDH is VIC / HEX / Yellow555 (533-580)
  • Analysis: Advanced Relative Quantification (C### samples used as calibrator for calculation of relative mCh signal)
cDNA Batch Pairing mCh mean Cp GAPDH (ref) mean Cp GAP-mCh delta Cp Verdict
K562_E001A1/A424.0584063323.539750060.698good mCh expression (switched E and C before)
K562_C001---28.9823552924.106640253.41E-02---
K562_E002B1/B424.024210723.574421680.7321---
K562_C001---28.0129655323.571072214.60E-02---
K562_E003C1/C429.09475970Invalidbad template
K562_C003---29.245795890Invalidbad template
SKNSH_E001D1/D419.6856271927.96917232311.6good mCh expression
SKNSH_C001---29.1806308525.689194428.89E-02good
SKNSH_E002E1/E417.2653377125.78400185366.8good mCh expression
SKNSH_C002---29.1505616124.96273355.49E-02good
SKNSH_E003F1/F417.7657160826.1942371344.5good mCh expression
SKNSH_C003---29.013944725.307929227.66E-02good
U2OS_E001G1/G428.7645563420.46642293.18E-03poor mCh signal, good GAPDH
U2OS_C001---27.8878763320.301466325.20E-03good GAPDH signal
U2OS_E002H1/H428.1951697722.820928152.41E-02poor mCh signal, good GAPDH
U2OS_C002---28.9995310819.691299371.58E-03good GAPDH signal


BAR CHART
RT-PCR results 05/16/14
Four each set of four bars:

  • 1 - blue = C### ratio = 2^(Cp GAPDH - Cp mCh)
  • 2 - red = Normalized C = C### ratio/ C### ratio
  • 3 - blue = E### ratio = 2^(Cp GAPDH - Cp mCh)
  • 4 - red = Normalized E = E### ratio/ C### ratio


CONCLUSIONS:

  • Continue transcript profiling with K562 E/C001, K562 E/C002, (throw out 003) all SK-N-SH samples
  • Try U2OS E/C002
  • Use stably transfected cell line KAH126 for further experiments, if needed



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