|
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:
- K562_E001 (E = experimental, +PcTF)
- K562_C001 (C = control, no PcTF)
- K562_E002
- K562_C002
- K562_E003
- K562_C003
- SKNSH_E001
- SKNSH_C001
- SKNSH_E002
- SKNSH_C002
- SKNSH_E003
- SKNSH_C003
- U2OS_E001
- U2OS_C001
- U2OS_E002
- 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
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
|
Pc-TF Genomics
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