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

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09/05/10

 * &#x2713; 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: > Templates
 * 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.)
 * 1) KAH126-1 -dox
 * 2) KAH126-1 + dox

--> Add 1.0 10 μM primers to 1-10 --> PCR (96-well)* (*Same as p16INK PCR, 8/26/10)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> 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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