cDNA Labeling Strategies

1
cDNA Labeling Strategies
Direct incorporaton of fluorescent nucleotides
2-step labeling method
Investigators who use the 2-step method report
that it reduces the bias associated with uneven
dye incorporation.
2
mRNA Amplification and Labeling
A A A A 3
5
T T T T- T7
Oligo(dT) primer
1st Strand cDNA synthesis
A A A A 3
5
T T T T- T7
2nd Strand cDNA synthesis
5
A A A A 3
T T T T- T7
cDNA purification
in vitro transcription
amino allyl UTP
Typical yields are 20mg amplified aRNA from 1-2
mg of total RNA. This is equivalent to a
200-500-fold amplification of mRNA.
UUUU 5
antisense RNA (aRNA)
aRNA purification
monoreactive Cy3 or Cy5
UUUU 5
Clean up labeled aRNA pool
UUUU 5
Combine Cy3 and Cy5 samples
UUUU 5
Hybridize to the microarray
UUUU 5