User:Karmella Haynes/Notebook/Polycomb project/2010/09/05

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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:

  1. FGF10 25A
  2. FGF10 25B
  3. ATM 26A
  4. ATM 26B
  5. BMP10 27A
  6. BMP10 27B
  7. HHEX 28A
  8. HHEX 28B
  9. MMP3 29A
  10. MMP3 29B
  11. mCherry f1/r1 (1:100 cDNA dln.)
  12. GAPDH 21A (1:1000 cDNA dln.)

> Templates

  1. KAH126-1 -dox
  2. 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)

RT-PCR 9/05/10

> 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)



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