User:Karmella Haynes/Notebook/PcTF Genomics/2014/05/14: Difference between revisions
From OpenWetWare
(11 intermediate revisions by the same user not shown) | |||
Line 15: | Line 15: | ||
---- | ---- | ||
'''RT-PCR: QC_plate, all cDNA batches'''<br> | '''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 | * 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: | * Experiment file name: DualColorProbe_UPL_Haynes051414 | ||
* Target gene wells: mCherry wells: 16x3 = 48; GAPD wells: 16x3 = 48 | * Target gene wells: mCherry wells: 16x3 = 48; GAPD wells: 16x3 = 48 | ||
* cDNA dilution: 1:1000 for all reactions | * cDNA dilution: 1:1000 for all reactions | ||
Line 23: | Line 24: | ||
cDNA Batches: | cDNA Batches: | ||
# K562_E001 | # K562_E001 (E = experimental, +PcTF) | ||
# K562_C001 | # K562_C001 (C = control, no PcTF) | ||
# K562_E002 | # K562_E002 | ||
# K562_C002 | # K562_C002 | ||
Line 33: | Line 34: | ||
# SKNSH_E002 | # SKNSH_E002 | ||
# SKNSH_C002 | # SKNSH_C002 | ||
# SKNSH_E003 | |||
# SKNSH_C003 | |||
# U2OS_E001 | # U2OS_E001 | ||
# U2OS_C001 | # U2OS_C001 | ||
# U2OS_E002 | # U2OS_E002 | ||
# U2OS_C002 | # U2OS_C002 | ||
Line 46: | Line 48: | ||
| align="center" style="background:#f0f0f0;"|'''(Single well)''' | | align="center" style="background:#f0f0f0;"|'''(Single well)''' | ||
| align="center" style="background:#f0f0f0;"|'''Gene Target mCh''' | | align="center" style="background:#f0f0f0;"|'''Gene Target mCh''' | ||
| align="center" style="background:#f0f0f0;"|'''Gene Target GAPD | | align="center" style="background:#f0f0f0;"|'''Gene Target GAPD''' | ||
|- | |- | ||
| 2x LC480 Probes Master||(7.5 μL)||360.0||360.0 | | 2x LC480 Probes Master||(7.5 μL)||360.0||360.0 | ||
Line 77: | Line 79: | ||
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 | |||
Revision as of 18:50, 16 May 2014
Pc-TF Genomics | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
|