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Application Note
Authors
Abstract
Introduction
Experimental
Sample
bp
12,216
5090
4072
3054
2036
1636
1018
506
220
Figure 1. Electrophoresis proles of amplied
products from 30 pg of reference DNA (1),
cell line NA03226 diluted to single cell levels (2),
a single cell (3), non-template control (4), and a
1Kb DNA ladder (L).
Microarray processing
Protocol optimization
Microarray specications
Design ID
21924
Total features
62,976
3,886
55,077
1,000
13
Unique probes
54,969 (99.8 %)
149 (0.27 %)
Pseudoautosomal probes
108
Exonic probes
14,259 (25.9 %)
Intragenic probes
36,995 (67.2 %)
Intergenic probes
18,082 (32.8 %)
33,307
Intergenic
78,946
CNV
26,688
Overall
41,448
54,455
15,553 (83.2 %)
3 probes
4,580 (24.5 %)
351 (97.0 %)
3 probes
226 (62.4 %)
DLRS
Signal intensity
Green
Red
Background noise
Green
Red
Signal-to-noise
Green
Red
Individual ref
1.66
224
413
32
44
0.78
318
366
10
43
37
0.78
324
373
53
48
NA03226: Chr. 9
Diluted gDNA
Non-amplied control
Chr. X
Single Cell
Non-amplied control
Figure 2. CN changes identied for samples hybridized to SurePrint G3 8x60K CGH microarrays for 16 hours.
(A) Detection of the expected CN gain on chromosome 9 of the amplied DNA from the cell line NA03226 (left) and in a non-amplied control gDNA (right).
(B) Sex determination from the X chromosome prole for a single cell (left) and in a non-amplied control female versus male hybridization (right). The extra
copy of the X chromosome indicates that the single cell is female.
Figure 3. Partial and whole chromosome aberrations identied in single cells genomes.
Conclusions
Using a straightforward and simple
process (Figure 4), genome-wide
CN changes of single cells can be
successfully proled with oligo-based
aCGH. Isolated single cells and
Single cell
WGA
WGA (8 reactions)
15 minutes
3 hours
10 minutes
2 hours 40 minutes
2 hours
15 to 16 hours
Washing
10 minutes
Scanning
15 minutes
20 minutes
Total
Figure 4. Workow for single cells analysis and the respective processing times estimated for 8 samples.
~ 24 hours
References
1. Munn et al. Improved detection
of aneuploid blastocysts using a
new 12-chromosome FISH test.
Reproductive Biomedicine Online,
2010, 20(1):92-97.
2. Barat-Houari et al. New multiplex
PCR-based protocol allowing indirect
diagnosis of FSHD on single cells: can
PGD be offered despite high risk of
recombination? European Journal of
Human Genetics, 2010, 18(5):533-538.
Part number
G5923A, Option 1
G2534A
Hybridization Oven
G2545A
G2530-60029
G4900DA
G1662AA
3. Bi et al. Detection of 1 Mb
microdeletions and microduplications
in a single cell using custom
oligonucleotide arrays. Prenatal
Diagnosis, 2012, 32(1):1020.
4. Cheng et al. Single-cell copy number
variation detection. Genome Biology,
2011, 12(8):R80.
5. Hellani et al. Successful pregnancies
after application of array-comparative
genomic hybridization in PGSaneuploidy screening. Reproductive
BioMedicine Online, 2008, 17(6):841847.
6. Traversa et al. The genetic screening
of preimplantation embryos by
comparative genomic hybridization.
Reproductive Biology, 2011, 11(3):51-60.
7. http://ccr.coriell.org/
www.genomics.agilent.com
This item is not approved for use in diagnostic
procedures. User is responsible for obtaining regulatory
approval or clearance from the appropriate authorities
prior to diagnostic use.
Information is subject to change without notice.
Agilent Technologies, Inc., 2012
Published in the USA, June 19, 2012
5991-0643EN