User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/14

From OpenWetWare

< User:Karmella Haynes | Notebook | PcTF Genomics | 2014 | 05(Difference between revisions)
Jump to: navigation, search
(Autocreate 2014/05/14 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
Current revision (21:50, 16 May 2014) (view source)
(05/14/14)
 
(16 intermediate revisions not shown.)
Line 8: Line 8:
-
==mm/dd/yy==
+
==05/14/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
-
* Line item 1
+
* RT-PCR: QC_plate, all cDNA batches
 +
 
----
----
-
'''Line item 1'''<br>
+
'''RT-PCR: QC_plate, all cDNA batches'''<br>
-
> Samples
+
* 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
-
{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
+
 
-
|-valign="top"
+
cDNA Batches:
-
| <u>Reagent</u> || <u>Volume</u>
+
# 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)
 +
{|
 +
| align="center" style="background:#f0f0f0;"|'''Reagent'''
 +
| align="center" style="background:#f0f0f0;"|'''(Single well)'''
 +
| align="center" style="background:#f0f0f0;"|'''Gene Target mCh'''
 +
| align="center" style="background:#f0f0f0;"|'''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)
 +
{|
 +
| align="center" style="background:#f0f0f0;"|'''Reagent'''
 +
| align="center" style="background:#f0f0f0;"|'''(Single well)'''
 +
| align="center" style="background:#f0f0f0;"|'''cDNA Template'''
 +
| align="center" style="background:#f0f0f0;"|'''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)
 +
 
 +
{|
 +
| align="center" style="background:#f0f0f0;"|'''cDNA Batch'''
 +
| align="center" style="background:#f0f0f0;"|'''Pairing'''
 +
| align="center" style="background:#f0f0f0;"|'''mCh mean Cp'''
 +
| align="center" style="background:#f0f0f0;"|'''GAPDH (ref) mean Cp'''
 +
| align="center" style="background:#f0f0f0;"|'''GAP-mCh delta Cp'''
 +
| align="center" style="background:#f0f0f0;"|'''Verdict'''
 +
|-
 +
| K562_E001 || A1/A4 || 36.416 || 30.876 || 2.15E-2 || bad mCh
 +
|-
 +
| 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 || 36.624 || 0 || invalid || not enough template
 +
|-
 +
| K562_C003 || --- || 34.329 || 0 || invalid || not enough template
 +
|-
 +
| SKNSH_E001 || D1/D4 || 20.812 || 29.718 || 480.1 || good mCh expression
 +
|-
 +
| SKNSH_C001 || --- || 33.691 || 30.259 || 9.27E-2 || good
 +
|-
 +
| SKNSH_E002 || E1/E4 || 20.810 || 29.964 || 569.5 || good mCh expression
 +
|-
 +
| SKNSH_C002 || --- || 32.599 || 29.739 || 0.1377 || good
|-
|-
-
| reagent 1 || # μL
+
| SKNSH_E003 || F1/F4 || 21.614 || 31.060 || 697.4 || good mCh expression
|-
|-
-
| reagent 2 || #
+
| SKNSH_C003 || --- || 36.165 || 30.564 || 2.06E-2 || good
|-
|-
-
| reagent 3 || #
+
| U2OS_E001 || G1/G4 || 34.713 || 24.584 || 8.93E-4 || poor mCh signal, good GAPDH
|-
|-
-
| reagent 4 || #
+
| U2OS_C001 || --- || 35.686 || 24.563 || 4.49E-4 || good GAPDH signal
|-
|-
-
| dH<sub>2</sub>O || #
+
| U2OS_E002 || H1/H4 || 34.961 || 24.539 || 7.29E-4 || poor mCh signal, good GAPDH
|-
|-
-
| &nbsp; || # μL
+
| U2OS_C002 || --- || 36.290 || 24.679 || 3.20E-4 || good GAPDH signal
|}
|}
-
--> Reaction conditions
+
Run whole plate again with 1:100 dilutions (10x more concentrated cDNA)
 +
BAR CHART<br>
 +
[[Image:DualColorProbe_UPL_Haynes051414_advrelquantChart.jpg|500px|RT-PCR results 05/14/14]]<br>
 +
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

Current revision

Pc-TF Genomics Main project page
Previous entry      Next entry


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:

  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) Gene Target mCh Gene Target GAPD
2x LC480 Probes Master(7.5 μL)360.0360.0
20 μM Forward primer(0.3 μL)14.414.4 GAPD primers
20 μM Reverse primer(0.3 μL)14.40
10 μM UPL probe(0.3 μL)14.414.4 GAPD probe
PCR H2O(0.1 μL)4.819.2
Total vol.(8.5 μL)408.0408.0


Template Master Mixes (16 tubes)

Reagent (Single well) cDNA Template no-template control
diluted Batch# cDNA(2.0 μL)12.00
PCR H2O(4.5 μL)27.00.0
Total vol.(6.5 μL)39.00.0


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 bad mCh
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 36.624 0 invalid not enough template
K562_C003 --- 34.329 0 invalid not enough template
SKNSH_E001 D1/D4 20.812 29.718 480.1 good mCh expression
SKNSH_C001 --- 33.691 30.259 9.27E-2 good
SKNSH_E002 E1/E4 20.810 29.964 569.5 good mCh expression
SKNSH_C002 --- 32.599 29.739 0.1377 good
SKNSH_E003 F1/F4 21.614 31.060 697.4 good mCh expression
SKNSH_C003 --- 36.165 30.564 2.06E-2 good
U2OS_E001 G1/G4 34.713 24.584 8.93E-4 poor mCh signal, good GAPDH
U2OS_C001 --- 35.686 24.563 4.49E-4 good GAPDH signal
U2OS_E002 H1/H4 34.961 24.539 7.29E-4 poor mCh signal, good GAPDH
U2OS_C002 --- 36.290 24.679 3.20E-4 good GAPDH signal

Run whole plate again with 1:100 dilutions (10x more concentrated cDNA)

BAR CHART
RT-PCR results 05/14/14
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



Personal tools