C-Fos Methylation
DNA Methylation sites in c-Fos promoter
Approximate MW:
38,337 daltons (unmethylated)
38,252 daltons (methylated)
each methylation site causes 15 dalton decrease in MW 
Rat c-fos Primers used by Alcala and Houpt
Primers designed with Primer 3 using the 300 bases upstream of c-Fos transcription start based on gi|62750806|ref|NC_005105.2|NC_005105 (equivalent to bases 172-471 of U02631, which claims start of 5’UTR is at 455.)
Forward at -261 (cFosSense-261):
- 5'-CCATCCCCGAAATCCTACAT-3'
Reverse at -6 (cFosAS-6):
- 5'-CAATCGCGGTTGGAGTAGTA-3'
Product size: 256 bp
PCR conditions for use with HotStarTaq mix (progam CFOSGENO on thermocycler)
95° for 15 min to activate Taq
Repeat 35 times:
- 94° for 1 min
- 59° for 1 min
- 72° for 1 min
- 72° for 10 min final extension
4° for overnight
Sequenced PCR product from Brain and Tail amplification on 1/29/09
Matched AY786174.1 base pairs 85-339
ccatcc ccgaaatcct acatgcggag ggtccaggag accttctaag atcccaattg tgaacactca taggtgaaag ttacagactg agacgggggt tgagagcctg gggcgtagag ttgatgacag ggagcccgca gagggcattc gggagcgctt tcccccctcc agtttctctg ttccgctcat gacgtagtaa gccattcaag cgcttctata aagcggccag ctgaggcgcc tactactcca accgcgatt
Rat c-fos Primers from Literature
From Shimizu et al., Cancer Sci 2007; 98: 1318–1322
Bisulfite sequencing (Genomic DNA)
GenBank AF126534
F: 5′-TATTTATAGGTGAAAGTTATAGATTG-3′
R: 5′-CACTAATAAAAACTACAAAACAAAACT-3′
Annealing temperature 54
Product size 292 bases
4-23-08 -- could NOT confirm with blast -- need to check earlier references
RT-PCR (from RNA)
GenBank X06769
F: 5′-TTGCGCAGATCTGTCCGTCT-3′
R: 5′-GTTGATCTGTCTCCGCTTGG-3′
Annealing temperature 65
4-23-08 -- confirmed with blast