User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/14: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==05/14/14== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * RT-PCR: QC_plate, all cDNA batches | ||
---- | ---- | ||
''' | '''RT-PCR: QC_plate, all cDNA batches'''<br> | ||
* 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) | |||
{| | |||
| 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 | |||
|- | |- | ||
| | | 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<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 | |||
Latest revision as of 23:58, 26 September 2017
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05/14/14
RT-PCR: QC_plate, all cDNA batches
Target Gene Primer/Probe Master mixes (2 tubes)
Run whole plate again with 1:100 dilutions (10x more concentrated cDNA) BAR CHART
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