Help- aCGH Agilent Arrays

[Helena Suarez]

Hi to all!
I'm new at aCGH Array and I need some advice.
I'm working following Agilent Oligonucleotide Array-Based CGHfor Genomic DNA Analysis using the Enzimatic Labeling protocol , from Agilent.

I'm starting from 500 ng of good quality gDNA and I perform a digestion and a labeling step as it is described on the protocol. The issue is that, when I mesure the degree of labeling and specific activity of the samples (labeled with Cy5) and the references (labeled with Cy3) I'm not sure if I'm getting good results...

The results I get are the following ones :

[B]Samples [ ] / Yield ug / pmol/ul /DOL (%) /sp.act
S1 (Cy5) = 196 ng/ul, 4.5ug, 5.9, 1.02%, 30.1
S2( Cy5)= 217,9 ng/ul, 5ug, 6.8, 1.06%, 31.2
R1 (Cy3)= 211,6 ng/ul, 4.4ug, 8.6, 1.38%, 40.6
R2 (Cy3)= 175,6 ng/ul, 3.68ug, 7.5, 1.45%, 42.71

And, on the protocol, it says that the values should be :

Tabla 27. Expected Yield and specific activity after labeling and clean-up (pagina 55 manual)

Input gDNa (ug) Yield (ug) sp.act Cy3 (pmol/ul) sp.act Cy5 (pmol/ul)
0,2-0,5/ 2,5-3/ 15-25/ 15-20
0,5/ 5-7/ 25-40/ 20-35
3/ 7-10/ 35-55/ 25-40

The concentrations ara Ok and I think that the pmol/ul are also ok, but the yield stil being a bit low and the specific activity of Cy3 seems to be low....any advice???
I would like to see some examples of values that have been used to hybridize and that have given good results. I don't know if i can hybridize these samples or not...


Thanks a lot in advance

[vix001]

Hi Helena,
We've run quite a lot of CGH arrays and tend to get specific activitys around 40 without any issues for running the arrays. We have had succesful data from yields and specific activities similar to yours so I wouldn't worry too much, the DNA quality seems to be the most important factor overall.
Here are some examples of our results;

DNA (ug) 1.5 Specific Activity Cy3 44.9 Specific Activity Cy5 56.6 Yield Cy3 2.5 Yield Cy5 3.4 DLRScore 0.19
DNA (ug) 1.5 Specific Activity Cy3 41.9 Specific Activity Cy5 55.7 Yield Cy3 2.6 Yield Cy5 3.4 DLRScore 0.19
DNA (ug) 1.3 Specific Activity Cy3 45.2 Specific Activity Cy5 69 Yield Cy3 2.8 Yield Cy5 3.9 DLRScore 0.21
DNA (ug) 1.5 Specific Activity Cy3 39 Specific Activity Cy5 59 Yield Cy3 3 Yield Cy5 4 DLRScore 0.17
DNA (ug) 1.5 Specific Activity Cy3 36 Specific Activity Cy5 52 Yield Cy3 6 Yield Cy5 3.5 DLRScore 0.16
DNA (ug) 1.5 Specific Activity Cy3 40 Specific Activity Cy5 48 Yield Cy3 8.1 Yield Cy5 5.6 DLRScore 0.17
DNA (ug) 2.2 Specific Activity Cy3 43.6 Specific Activity Cy5 67.4 Yield Cy3 8.5 Yield Cy5 7.4 DLRScore 0.26

I hope this helps,

Vicky