09/05/10
- ✓ RT-PCR: confirm Nanostring gene candidate set for KAH126-1 -/+ dox (test new primers)
RT-PCR
> Test new primers for Nanostring gene candidate set for KAH126-1 -/+ dox (test new primers)
> Use cDNA from Nanostring samples
> Same PCR conditions as p16 (see 8/26/10)
> Primers:
- FGF10 25A
- FGF10 25B
- ATM 26A
- ATM 26B
- BMP10 27A
- BMP10 27B
- HHEX 28A
- HHEX 28B
- MMP3 29A
- MMP3 29B
- mCherry f1/r1 (1:100 cDNA dln.)
- GAPDH 21A (1:1000 cDNA dln.)
> Templates
- KAH126-1 -dox
- KAH126-1 + dox
Reagent |
1-10, MM (x10) |
11 |
12
|
cDNA |
5.0 (1:1) |
5.0 (1:1000) |
0.5 (1:1000)
|
10 μM primers |
--- |
1.0 |
1.0
|
2x GoTaq Green |
100.0 |
10.0 |
10.0
|
dH2O |
85.0 |
3.5 |
8.5
|
|
20.0 μL |
|
|
--> Add 1.0 10 μM primers to 1-10
--> PCR (96-well)*
- 95°C/ 3 min.
- [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
- 72°C/ 3 min.
- 4°C/ ∞
(*Same as p16INK PCR, 8/26/10)
> Conclusions: Results do not look great. Re-evaluated Nanostring data to filter out genes that have a high basal level of expression and/or change in the negative controls. Included genes that showed at least a 2-fold change in KAH126-1, 128-8.3, and/or 129-4.
> Design new primers for this sub-set
- CDKN2A (done)
- HHEX (test primers on KAH129-4, Nanostring showed >2-fold increase)
- IL4 (new)
- MMP12 (new)
- NPPA (new)
- THPO (new)
- TNF (new)
|