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PCR Applications Guide | 61

Gene Expression

Figure 6.3 illustrates the determination of ERBB2 transcript levels relative to the EEF2

reference gene. The “best coverage” ERBB2 assay was tested with four fresh-frozen breast

tumor RNA samples (OriGene Technologies, Inc). FAM (ERBB2) concentration, VIC (EEF2)

concentration, and normalized ERBB2 to EEF2 concentrations are shown in Figure 6.3

(blue, green, and maroon, respectively).

Fig. 6.3. Concentrations of ERBB2 and EEF2 gene expression.

CR560536 cD CR560258 cD CR562124 cD CR561507 cD

Sample

Concentration, copies/µl

Normalized ERBB2:EEF2

4,000

3,200

2,400

1,600

800

3,400

215

0.0633

1.28

658

514

1,650

1.57

1,050

1,030

3.04

3,130

Figure 6.4 shows a twofold change in sample concentration that demonstrates linearity

of the assay spanning two log orders.

Fig. 6.4. Twofold dilution series demonstrating linearity across two log orders.

Sample

Concentration, copies/µl

Normalized ERBB2:EEF2

1,000

100

0.1

0.0

2.5

3,120

2,500

2.49

1,550

1,250

2.45

775

634

2.47

395

321

2.41

2.51

2.31

2.37

2.28

193

160

96.5

76.9

45.9

39.8

24.1

20.3

12.1

10.7

CR561507 cDN