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 Pc-TF Genomics
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01/27/14
- qRT-PCR experiment - PlateK562_1/ U2OS
- U2OS RT-PCR - RNA extraction & cDNA synthesis
RNA Extraction
- Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit.
Samples (TRIzol-lysed U2OS samples from -80°C freezer)
- Tube 1-A (+PcTF)
- Tube 1-A (+PcTF)
- Tube 4-A (mock)
- Tube 4-A (mock)
Procedure notes (based on Carly's notes from 1/10/13)
TRIzol steps
- Clean all work surfaces and pipettors with RNase-zap.
- Thaw TRIzol-lysed cells at room temp
- Under fume hood: Add 100 μL chloroform to each sample
- Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min.
- Centrifuge in cold room for 15 min @ 12,000G
- Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE)
- Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. Mix and incubate at room temp (refer to RNeasy protocol)
Spin-column steps
- Open up one pink, sealed spin-column (with attached cap) per sample, label it, and place into collection tube(s).
- Transfer RNA/ethanol mix to each spin column.
- Spin tubes at top speed for 30 sec (room temp).
- Discard flow-through liquid and add 700μL of RW1 Buffer to the spin column.
- Spin tubes at top speed for 30 sec (room temp).
- Put collection vial in a fresh collection tube and discard the old one.
- --> Add 500μL of RBE Buffer to the spin column.
- Spin tubes at top speed for 30 sec (room temp) then discard the liquid flow-through.
- Repeat the above two steps again. -->
- Discard the flow-through and spin again.
- Transfer liquid to a fresh 1.5 mL tube (RNase-free) and add 30 μL of RNase-free H2O.
- Let tubes sit for 1 min at room temp.
- Spin tubes for 1 min (room temp).
- Store at -80°C or proceed to RNA concentration measurement.
cDNA Synthesis
- Use Superscript III kit (freezer)
Procedure notes
- Kit components kept on ice: RNase H, SS III RT, and RNase out
- Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
qRT-PCR - Plate K562_1/ U2OS
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Template
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Gene Target
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| Rxn 1: |
treated cells |
KIT, primers 13/14
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| Rxn 2: |
treated cells |
TNFRSF11A, primers 15/16
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| Rxn 3: |
treated cells |
EGFR, primers 17/18
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| Rxn 4: |
treated cells |
WT1, primers 19/20
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| Rxn 5: |
treated cells |
HLF, primer 21/22
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| Rxn 6: |
treated cells |
BCL6, primer 23/24
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| Rxn 7: |
treated cells |
mCh
|
| Rxn 8: |
treated cells |
ref. gene, GAPD
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| Rxn 9: |
untreated cells |
KIT, primers 13/14
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| Rxn 10: |
untreated cells |
TNFRSF11A, primers 15/16
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| Rxn 11: |
untreated cells |
EGFR, primers 17/18
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| Rxn 12: |
untreated cells |
WT1, primers 19/20
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| Rxn 13: |
untreated cells |
HLF, primer 21/22
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| Rxn 14: |
untreated cells |
BCL6, primer 23/24
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| Rxn 15: |
untreated cells |
mCh
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| Rxn 16: |
untreated cells |
ref. gene, GAPD
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| Rxn 17: |
no template |
KIT, primers 13/14
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| Rxn 18: |
no template |
TNFRSF11A, primers 15/16
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| Rxn 19: |
no template |
EGFR, primers 17/18
|
| Rxn 20: |
no template |
WT1, primers 19/20
|
| Rxn 21: |
no template |
HLF, primer 21/22
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| Rxn 22: |
no template |
BCL6, primer 23/24
|
| Rxn 23: |
no template |
mCh
|
| Rxn 24: |
no template |
ref. gene, GAPD
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Primer/Probe Master Mixes (8 total)
| Reagent
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(Single well)
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Gene Target 1 - 7 (x10)
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Gene Target GAPD (x10)
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| 2x LC480 Probes Master |
(7.5 μL) |
75 |
75
|
| 20 μM Forward primer |
(0.3 μL) |
3 |
3.0 GAPD primers*
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| 20 μM Reverse primer |
(0.3 μL) |
3 |
---
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| 10 μM UPL probe |
(0.3 μL) |
3 |
3.0 GAPD UPL probe*
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| PCR H2O |
(0.1 μL) |
1 |
4
|
| Total vol. |
(8.5 μL) |
85 |
85
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Template Master Mixes
- Use 1:10 dilution of cDNA for targets
- Use 1:100 dilution of cDNA for GAPD
| Reagent
|
(Single well)
|
treated cDNA Template (x25)
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untreated cDNA Template (x25)
|
no Template (x25)
|
| diluted cDNA |
(2.0 μL) |
50 |
50 |
---
|
| PCR H2O |
(4.5 μL) |
112.5 |
112.5 |
162.5
|
| Total vol. |
(6.5 μL) |
162.5 |
162.5 |
162.5
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96-well plate
- First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM
- Transfer 15 μL from well 1 to wells 2 and 3 for each set
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Pc-TF Genomics
