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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngPc-TF Genomics Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


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) 363.75 363.75
20 μM Forward primer (0.3 μL) 14.55 14.55 GAPD primers
20 μM Reverse primer (0.3 μL) 14.55 0
10 μM UPL probe (0.3 μL) 14.55 14.55 GAPD probe
PCR H2O (0.1 μL) 4.85 19.40
Total vol. (8.5 μL) 412.25 412.25

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_E001 A1/A4 24.05840633 23.53975006 0.698 good mCh expression (switched E and C before)
K562_C001 --- 28.98235529 24.10664025 3.41E-02 ---
K562_E002 B1/B4 24.0242107 23.57442168 0.7321 ---
K562_C001 --- 28.01296553 23.57107221 4.60E-02 ---
K562_E003 C1/C4 29.0947597 0 Invalid bad template
K562_C003 --- 29.24579589 0 Invalid bad template
SKNSH_E001 D1/D4 19.68562719 27.96917232 311.6 good mCh expression
SKNSH_C001 --- 29.18063085 25.68919442 8.89E-02 good
SKNSH_E002 E1/E4 17.26533771 25.78400185 366.8 good mCh expression
SKNSH_C002 --- 29.15056161 24.9627335 5.49E-02 good
SKNSH_E003 F1/F4 17.76571608 26.1942371 344.5 good mCh expression
SKNSH_C003 --- 29.0139447 25.30792922 7.66E-02 good
U2OS_E001 G1/G4 28.76455634 20.4664229 3.18E-03 poor mCh signal, good GAPDH
U2OS_C001 --- 27.88787633 20.30146632 5.20E-03 good GAPDH signal
U2OS_E002 H1/H4 28.19516977 22.82092815 2.41E-02 poor mCh signal, good GAPDH
U2OS_C002 --- 28.99953108 19.69129937 1.58E-03 good 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