01/27/14
- qRT-PCR experiment - PlateK562_1/ U2OS
- U2OS RT-PCR - RNA extraction & cDNA synthesis
RNA Extraction
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
- Open up pink, sealed spin-columns
- 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.
- Transfer to collection tube (we have K562 1, K562 2, SK-N-SH 1, and SK-N-SH 2)
- Spin tubes at top speed for 30 sec
- Discard collected liquid and add 700μL of RW1 Buffer
- Spin tubes at top speed for 30 sec
- Put collection vial in a fresh collector tube and discard of the old one
- Add 500μL of RBE Buffer
- Spin tubes at top speed for 30 sec then discard of the liquid waste
- Repeat the above two steps again
- Discard of the waste and spin again
- Transfer liquid to a fresh tube and add 30mL of RNase-Free H20
- Let tubes sit for 1 min
- Spin tubes for 1 min
- Store at -80°C
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
|
Template
|
Gene Target
|
Rxn 1: |
treated cells |
KIT, primers 13/14
|
Rxn 2: |
treated cells |
TNFRSF11A, primers 15/16
|
Rxn 3: |
treated cells |
EGFR, primers 17/18
|
Rxn 4: |
treated cells |
WT1, primers 19/20
|
Rxn 5: |
treated cells |
HLF, primer 21/22
|
Rxn 6: |
treated cells |
BCL6, primer 23/24
|
Rxn 7: |
treated cells |
mCh
|
Rxn 8: |
treated cells |
ref. gene, GAPD
|
Rxn 9: |
untreated cells |
KIT, primers 13/14
|
Rxn 10: |
untreated cells |
TNFRSF11A, primers 15/16
|
Rxn 11: |
untreated cells |
EGFR, primers 17/18
|
Rxn 12: |
untreated cells |
WT1, primers 19/20
|
Rxn 13: |
untreated cells |
HLF, primer 21/22
|
Rxn 14: |
untreated cells |
BCL6, primer 23/24
|
Rxn 15: |
untreated cells |
mCh
|
Rxn 16: |
untreated cells |
ref. gene, GAPD
|
Rxn 17: |
no template |
KIT, primers 13/14
|
Rxn 18: |
no template |
TNFRSF11A, primers 15/16
|
Rxn 19: |
no template |
EGFR, primers 17/18
|
Rxn 20: |
no template |
WT1, primers 19/20
|
Rxn 21: |
no template |
HLF, primer 21/22
|
Rxn 22: |
no template |
BCL6, primer 23/24
|
Rxn 23: |
no template |
mCh
|
Rxn 24: |
no template |
ref. gene, GAPD
|
Primer/Probe Master Mixes (8 total)
Reagent
|
(Single well)
|
Gene Target 1 - 7 (x10)
|
Gene Target GAPD (x10)
|
2x LC480 Probes Master |
(7.5 μL) |
75 |
75
|
20 μM Forward primer |
(0.3 μL) |
3 |
3.0 GAPD primers*
|
20 μM Reverse primer |
(0.3 μL) |
3 |
---
|
10 μM UPL probe |
(0.3 μL) |
3 |
3.0 GAPD UPL probe*
|
PCR H2O |
(0.1 μL) |
1 |
4
|
Total vol. |
(8.5 μL) |
85 |
85
|
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)
|
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
|
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
|