User manual

60 | Droplet Digital
™
PCR Applications Guide
Gene Expression
Fig. 6.2. Comparison of one-step RT-ddPCR and two-step RT-ddPCR with in vitro transcribed RNAs
spiked in Ambion human brain reference RNA.
ddPCR Gene Expression Data
As shown in Figure 6.2, cDNA copies were made on three in vitro transcribed RNAs with
the one-step RT-ddPCR kit for probes or two-step RT-qPCR with iScript advanced cDNA
synthesis kit for RT-qPCR. The data are generally comparable on most of the assay
locations. A few sites are much lower in one-step RT-ddPCR because those sites may
be involved in secondary structures and the priming sites might not be accessible to the
reverse transcription enzyme. You must decide whether one-step or two-step RT-ddPCR
is best for your experiment.
ALB_1_2
800
700
600
500
400
300
200
100
0
ALB_2_3
ALB_3_4
ALB_5_6
ALB_6_7
ALB_7_8
ALB_8_9
ALB_9_10
ALB_10_11
ALB_11_12
ALB_12_13
ALB_13_14
ALB_14_15
ALAS_3_4
ALAS_4_5
AL AS_7_8
ALAS_9_10
AL AS_10_11
AL AS_11_12
LAG_1_2
LAG_3_4
LAG_4_5
LAG_5_6
L AG _7_ 8
Bio-Rad iScript advanced cDNA synthesis
kit for RT-qPCR
Bio-Rad one-step RT-ddPCR kit for probes
Sample
HER2 Study
The human epidermal growth factor receptor 2 gene (HER2) is encoded by the ERBB2
proto-oncogene and is involved in signal transduction for cell growth and differentiation.
It is a cell surface receptor for tyrosine kinase and a proto-oncogene. Overexpression of
ERBB2 is of clinical relevance in breast cancer because of its prognostic value in correlating
elevated expression with worsening clinical outcome. ERBB2 assessment is also important
because successful antitumor treatment with Herceptin is strongly correlated with ERBB2
overexpression in the tumor (about 30% of all breast tumors overexpress ERBB2).
Data Analysis Results
Results demonstrate that ddPCR can be used to assess expression levels of HER2 transcripts
in FFPE human breast tissues samples that were concurrently assessed via pathologists using
fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) methods.
Concentration, copies/µl