Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/02/19

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Summary

Having received a cDNA duplicate of our samples submitted to DNASU for RNA-seq, I'm going to do a test run on the qPCR light cycler using mCherry and GAPD as targets.



Goals

  • ✓ qPCR test run
  • ✓ Passage K562



Protocol

Universal Probe Library Assay (qPCR)

passaging cells




Notes

Templates:

  • KAH126/U2OS Well 1 (+ dox)
  • KAH126/U2OS Well 2 (+ dox)
  • KAH126/U2OS Well 4 (- dox)
  • U2OS + PcTF-RFP (transiently transfected)
  • U2OS (no transfection control)
  • SKNSH + PcTF-RFP (transiently transfected)
  • SKNSH (no transfection control)
  • no template control

Targets:

  • mCherry
  • GAPD

Primer/probe master mixes:

Reagent (Single well) Target Gene (x24.5) Roche GAPD (x24.5)
2x LC480 Probes Master (7.5 μL) 183.75 183.75
20 μM Forward primer (0.3 μL) 7.35 7.35 GAPD primers
20 μM Reverse primer (0.3 μL) 7.35 0.00
10 μM UPL probe (0.3 μL) 7.35 7.35 GAPD probe
PCR H2O (0.1 μL) 2.45 9.80
Total vol. (8.5 μL) 208.25 208.25

Template master mixes:

Reagent (Single well) cDNA Template (x6.5) no-template control (x6.5)
diluted Batch# cDNA (2.0 μL) 13.0 0.00
PCR H2O (4.5 μL) 29.3 42.25
Total vol. (6.5 μL) 42.3 42.25

Reaction master list:

Rxn # Template Gene Target
Rxn 1: U2OS (no transfection control) mCherry
Rxn 2: U2OS (no transfection control) mCherry
Rxn 3: U2OS (no transfection control) mCherry
Rxn 4: U2OS (no transfection control) GAPD
Rxn 5: U2OS (no transfection control) GAPD
Rxn 6: U2OS (no transfection control) GAPD
Rxn 7: U2OS + PcTF-RFP (transiently transfected) mCherry
Rxn 8: U2OS + PcTF-RFP (transiently transfected) mCherry
Rxn 9: U2OS + PcTF-RFP (transiently transfected) mCherry
Rxn 10: U2OS + PcTF-RFP (transiently transfected) GAPD
Rxn 11: U2OS + PcTF-RFP (transiently transfected) GAPD
Rxn 12: U2OS + PcTF-RFP (transiently transfected) GAPD
Rxn 13: SKNSH (no transfection control) mCherry
Rxn 14: SKNSH (no transfection control) mCherry
Rxn 15: SKNSH (no transfection control) mCherry
Rxn 16: SKNSH (no transfection control) GAPD
Rxn 17: SKNSH (no transfection control) GAPD
Rxn 18: SKNSH (no transfection control) GAPD
Rxn 19: SKNSH + PcTF-RFP (transiently transfected) mCherry
Rxn 20: SKNSH + PcTF-RFP (transiently transfected) mCherry
Rxn 21: SKNSH + PcTF-RFP (transiently transfected) mCherry
Rxn 22: SKNSH + PcTF-RFP (transiently transfected) GAPD
Rxn 23: SKNSH + PcTF-RFP (transiently transfected) GAPD
Rxn 24: SKNSH + PcTF-RFP (transiently transfected) GAPD
Rxn 25: KAH126/U2OS Well 4 (- dox) mCherry
Rxn 26: KAH126/U2OS Well 4 (- dox) mCherry
Rxn 27: KAH126/U2OS Well 4 (- dox) mCherry
Rxn 28: KAH126/U2OS Well 4 (- dox) GAPD
Rxn 29: KAH126/U2OS Well 4 (- dox) GAPD
Rxn 30: KAH126/U2OS Well 4 (- dox) GAPD
Rxn 31: KAH126/U2OS Well 2 (+ dox) mCherry
Rxn 32: KAH126/U2OS Well 2 (+ dox) mCherry
Rxn 33: KAH126/U2OS Well 2 (+ dox) mCherry
Rxn 34: KAH126/U2OS Well 2 (+ dox) GAPD
Rxn 35: KAH126/U2OS Well 2 (+ dox) GAPD
Rxn 36: KAH126/U2OS Well 2 (+ dox) GAPD
Rxn 37: KAH126/U2OS Well 1 (+ dox) mCherry
Rxn 38: KAH126/U2OS Well 1 (+ dox) mCherry
Rxn 39: KAH126/U2OS Well 1 (+ dox) mCherry
Rxn 40: KAH126/U2OS Well 1 (+ dox) GAPD
Rxn 41: KAH126/U2OS Well 1 (+ dox) GAPD
Rxn 42: KAH126/U2OS Well 1 (+ dox) GAPD
Rxn 43: no template control mCherry
Rxn 44: no template control mCherry
Rxn 45: no template control mCherry
Rxn 46: no template control GAPD
Rxn 47: no template control GAPD
Rxn 48: no template control GAPD

Plate layout:

1 2 3 4 5 6 7 8 9 19 11 12
A Rxn 1 Rxn 2 Rxn 3 Rxn 4 Rxn 5 Rxn 6
B Rxn 7 Rxn 8 Rxn 9 Rxn 10 Rxn 11 Rxn 12
C Rxn 13 Rxn 14 Rxn 15 Rxn 16 Rxn 17 Rxn 18
D Rxn 19 Rxn 20 Rxn 21 Rxn 22 Rxn 23 Rxn 24
E Rxn 25 Rxn 26 Rxn 27 Rxn 28 Rxn 29 Rxn 30
F Rxn 31 Rxn 32 Rxn 33 Rxn 34 Rxn 35 Rxn 36
G Rxn 37 Rxn 38 Rxn 39 Rxn 40 Rxn 41 Rxn 42
H Rxn 43 Rxn 44 Rxn 45 Rxn 46 Rxn 47 Rxn 48



Results/Conclusions

qPCR results:

Pairing Sample Name Targets References Mean Cp Mean Cp Target/Ref Target/Ref Error Normalized Normalized Error
U2OS_C003 mCherry GAPD 29.02218992 16.332044 1.51E-04 1.10E-05 1
B1/B4 U2OS_E003 mCherry GAPD 17.44978164 18.23301659 1.721 0.2379 1.14E+04 1697
SKNSH_C004 mCherry GAPD 29.96936898 18.03409171 2.55E-04 3.69E-05 1
D1/D4 SKNSH_E004 mCherry GAPD 17.00077371 17.76315881 1.696 0.1696 6643 1116
U2OSKAH126_C001 mCherry GAPD 21.19016797 16.29073099 3.35E-02 9.48E-04 1
F1/F4 U2OSKAH126_E001_B mCherry GAPD 15.37687163 16.5109341 2.195 0.2424 65.5 7.09
G1/G4 U2OSKAH126_E001_A mCherry GAPD 15.11221216 16.37021015 2.392 0.2268 71.38 6.706

Overall, looks pretty good. Cp values are very high though, because I forgot to dilute the cDNA before running the experiment. Will have to try again with 1:100 or 1:1000 dilution. Check with Dr. Haynes about exact amount of dilution.