Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 237

18.3.3 Confirmation of Dystroglycan Knockdown
by qRT-PCR Analysis

To further confirm that siRNA injection specifically knocked down dystroglycan
gene expression, we also performed real-time qRT-PCR. Briefly, total RNA was
isolated from uninjected control, KD control, andMD zebrafish at 4, 6, and 20 hpi and
qRT-PCR was performed using conventional methods. Results showed that dystro-
glycan gene expression (expressed as percent of uninjected control) decreased in MD
zebrafish at 4, 6, and 20 hpi (85.1 3.2%, respec-

tively), confirming dystroglycan knockdown and results by immunostaining
(Fig. 18.3).

8.0%, 94.2 8.4%, and 59.5

18.3.4 MD Zebrafish Exhibit Short, Disorganized
Myotomes

MD zebrafish exhibited short myotomes lacking the characteristic chevron, V-shaped
structure (Fig. 18.2, red arrow; thick black outline) (Parsons et al., 2002). In contrast,
KD control zebrafish exhibited normal V-shaped myotomes (Fig. 18.2, black arrow;
thick black outline). In MD zebrafish, some myotomes were disrupted or missing.
Myotomes in the tail are primarily comprised of muscle fibers that align along the
anterior-posterior axis of the body and tail length reflects muscle fiber length.
Next, we measured eight myotomes along the anterior-posterior body axis (two
anterior and six posterior to the anal pore) (blue lines, Fig. 18.2). A significant
difference was observed ( P ¼7.110 14 ); mean length for KD control zebrafish was
0.642 0.075 mm and for MD zebrafish was 0.450 0.079mm ( n ¼ 33), which was

30% shorter ((1 0.450/0.642) 100% ¼ 29.9%) than KD control (Fig. 18.4).

0.800

KD controls
MD

0.700

0.600

0.500

0.400

0.300

0.200

0.100

0.000

Figure 18.4 Decreased myotome length in MD zebrafish. Myotomes (eight myotomes) in
3dpf MD zebrafish were 39% shorter than KD control ( P 10 14 , n

¼ 7.1 ¼ 33).