05/14/14
- RT-PCR: QC_plate, all cDNA batches
RT-PCR: QC_plate, all cDNA batches
- 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_Haynes051414
- Target gene wells: mCherry wells: 16x3 = 48; GAPD wells: 16x3 = 48
- cDNA dilution: 1:1000 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)
|
Gene Target mCh
|
Gene Target GAPD
|
2x LC480 Probes Master |
(7.5 μL) |
360.0 |
360.0
|
20 μM Forward primer |
(0.3 μL) |
14.4 |
14.4 GAPD primers
|
20 μM Reverse primer |
(0.3 μL) |
14.4 |
0
|
10 μM UPL probe |
(0.3 μL) |
14.4 |
14.4 GAPD probe
|
PCR H2O |
(0.1 μL) |
4.8 |
19.2
|
Total vol. |
(8.5 μL) |
408.0 |
408.0
|
Template Master Mixes (16 tubes)
Reagent
|
(Single well)
|
cDNA Template
|
no-template control
|
diluted Batch# cDNA |
(2.0 μL) |
12.0 |
0
|
PCR H2O |
(4.5 μL) |
27.0 |
0.0
|
Total vol. |
(6.5 μL) |
39.0 |
0.0
|
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 |
36.416 |
30.876 |
2.15E-2 |
---
|
K562_C001 |
--- |
28.381 |
29.313 |
1.907 |
---
|
K562_E002 |
B1/B4 |
27.404 |
27.596 |
1.142 |
---
|
K562_C001 |
--- |
35.233 |
29.435 |
1.80E-2 |
---
|
K562_E003 |
C1/C4 |
### |
### |
---
|
K562_C003 |
--- |
### |
### |
---
|
SKNSH_E001 |
D1/D4 |
### |
### |
---
|
SKNSH_C001 |
### |
### |
---
|
SKNSH_E002 |
### |
### |
---
|
SKNSH_C002 |
### |
### |
---
|
SKNSH_E003 |
### |
### |
---
|
SKNSH_C003 |
### |
### |
---
|
U2OS_E001 |
### |
### |
---
|
U2OS_C001 |
### |
### |
---
|
U2OS_E002 |
### |
### |
---
|
U2OS_C002 |
### |
### |
---
|
Run whole plate again with 1:100 dilutions
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
|