User manual

62 | Droplet Digital
™
PCR Applications Guide
Gene Expression
Figure 6.5 is an example of normal breast tissue compared with HER2+ tissues
(fresh frozen).
Fig. 6.5. Comparison of HER2 expression levels of fresh frozen tumor versus normal tissue.
CR560536 cD CR560258 cD CR562124 cD CR561507 cD Ambion normal
Sample
Concentration, copies/µl
1,000
100
10
1.0
0.10
0.01
1.0
0.10
0.01
618
217
0.0636
1.27
3,14 0
656
3,410
1,730
1,090
1.58
3.03
1,040
1,170
15.7
0.0133
Normalized ERBB2 to GAPDH concentration
Figure 6.6 shows the quantification of RNA transcript levels compared with DNA copy
number. Commercial (Applied Biosystems) ERBB2, GAPDH-PL, and EEF2 gene expression
TaqMan assays were tested with breast tumor FFPE samples. ddPCR was used to
determine the amounts of transcripts in a subset of 12 samples (from the genomic copy
number study above) that are normalized to commonly expressed genes GAPDH and EEF2.
Fig. 6.6. Rank ordering of gene expression levels in 12 clinical samples and genomic copy number
status of ERBB2.
60
50
40
30
20
10
0
ddPCR: DNA CNV
RNA ERBB2-B+3'/EEF2
38 32 40 34 37 45 41 30 43 49 28 47
Sample number
Normalized transcript level
Her2– Her2+
RNA ERBB2-B+3'/GAPDH -PL
When we studied the clinical FFPE samples using ddPCR and standard FISH and IHC
methods, our results demonstrated that ddPCR can rank order the samples in complete
agreement with the current standard methods and that ddPCR has the added benefit of
providing quantitative results.