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Droplet Digital

™

PCR Applications Guide | 63

Gene Expression

Table 6.1 shows a comparison of IHC, FISH, genomic copy number, and transcript level in

clinical samples. The table shows the capability of ddPCR to determine gene copies and

ERBB2 cDNA transcript levels when normalized to a reference transcript (EEF2 or GAPDH).

ddPCR results are in excellent agreement with pathology results, and have the added

beneﬁt of being quantitative.

Table 6.1. Comparison of clinical pathology results with ddPCR gene expression data.

Clinical Pathology ddPCR: CNVs or Normalized RNA Levels

Sample

Number

Year Specimen

Taken

HER2 IHC

Score

FISH HER2/

CEP17 Ratio

DNA

HER2/

CEP17

RNA

ERRB2-

B+3'/EEF2

RNA

ERRB2-

B+3'/GAPDH

38 2009 1+ 0.8 2.20 0.22 0.27

32 2010 1+ 1.2 1.56 0.29 0.46

40 2008 2+ Negative 2.46 0.54 0.88

34 2009 1+ Not done 2.61 0.17 1.42

37 2008 0 Not done 1.96 0.98 1.54

45 2006 Negative Not done 2.55 0.63 1.89

41 2008 1+ (resection) Negative 2.53 0.85 3.01

30 2009 1+ Not done 2.37 0.86 3.87

43 2007 2+ Positive 4.76 2.97 8.31

49 2006 3+ Not done 4.15 1.79 11.3

28 2010 3+ Not done 28.0 33.9 29.1

47 2006 3+ Not done 28.4 33.6 33.7

CNV, copy number variation; FISH, ﬂuorescence in situ hybridization; IHC, immunohistochemistry.

ddPCR provides the advantage of accurate and precise measurements of transcript levels.

It also provides accurate genomic copy number determinations of samples with low-

and high-order copy changes, determined by the ratio of the target concentration to the

reference concentration. Important considerations for ddPCR applied to gene expression

studies include the type of method used to obtain cDNA, one-step versus two-step reverse

transcription, and optimizing assays for maximum performance.