Fig. 1Representative photos of Western blot analysis. (A) HPRT1 exhibits reproducibility in the cerebrum, cerebellum, spleen, but shows notable variation in the aorta. (B) GYS1 exhibits reproducibility in the cerebrum, cerebellum and femoral muscle, but it shows only faint bands in the liver, spleen, and kidney. (C) ACTB exhibits relatively reproducible expression level in the cerebrum, cerebellum, atrium, liver and kidney. Unlike other housekeeping genes, ACTB exhibits reproducibility in the aorta (individual rat numbers are shown as case no. 1 through no. 8).
Fig. 2Expression stability of each gene represents as coefficient of variation (CV) value across tissue types. (A) HPRT1 shows stability in order of the spleen, kidney, cerebrum, cerebellum, ventricle, and femoral muscle, but a remarkably high CV value in the aorta. (B) PPIA shows the highest stability in the cerebellum. (C) With GYS1, all organ tissues are found to have CV values over 10%. The femoral muscle is relatively stable compared to other genes. (D) With TBP, all organ tissues are found to have CV values over 10%. (E) YWHAZ exhibits the highest stability in cerebellum, whereas it shows prominently high CV values in the femoral muscle and aorta. (F) GAPDH demonstrates the highest stability in the cerebrum, but significant variance in the aorta. (G) With ACTB, all organ tissues are found to have CV values over 10%. However, the kidney is relatively stable compared to other genes. Femoral m., femoral muscle; psoas m., Psoas muscle.
Fig. 3Comparison of expression stability between genes in each tissue type. (A) In the cerebrum, GAPDH demonstrates the highest stability, while GYS1 shows the least stable expression. (B) In the cerebellum, PPIA shows the highest stability, while TBP was the least stable. (C) In the ventricle, GAPDH exhibits the highest stability, while TBP was the least stable. (D) In the atrium, all seven genes are found to have coefficient of variation (CV) values over 10%. However, PPIA is relatively stable compared to other genes. (E) In psoas muscle, GAPDH shows the highest stability. (F) In femoral muscle, HPRT1 exhibits the highest stability. YWHAZ and ACTB demonstrate the highest variability in the femoral muscle. (G) In the liver, all genes are found to have CV values over 10%. However, ACTB is relatively stable compared to other genes. (H) In the spleen, HPRT1 demonstrates the highest stability. (I) In the kidney, PPIA shows the highest stability. (J) In aorta, all seven genes demonstrate high CV values over 10%.
Table 1.List of antibodies used
Antibody |
Molecular weight (kDa) |
Company |
Dilution |
Organ(s) |
HPRT1
|
24 |
Abcam |
1:16,000 |
Psoas muscle, femoral muscle |
|
|
|
1:32,000 |
Cerebrum, cerebellum, ventricle, atrium, aorta, liver, kidney, spleen |
PPIA
|
18 |
Cell Signaling |
1:1,000 |
Ventricle, atrium, liver, kidney, spleen |
|
|
|
1:8,000 |
Cerebrum, cerebellum, aorta, psoas muscle, femoral muscle |
GYS1
|
85 |
Abcam |
1:1,000 |
Cerebrum, cerebellum, aorta, liver, kidney, spleen |
|
|
|
1:8,000 |
Ventricle, atrium, psoas muscle, femoral muscle |
TBP
|
38 |
Abcam |
1:1,000 |
Ventricle, atrium, liver, kidney, spleen, cerebrum, cerebellum, aorta, psoas muscle, and femoral muscle |
YWHAZ
|
30 |
Millipore |
1:4,000 |
Ventricle, atrium, psoas muscle, femoral muscle |
|
|
|
1:8,000 |
Aorta, liver, kidney, spleen |
|
|
|
1:16,000 |
Cerebrum, cerebellum |
GAPDH
|
37 |
Cell Signaling |
1:64,000 |
Ventricle, atrium, liver, kidney, spleen, cerebrum, cerebellum, aorta, psoas muscle, femoral muscle |
ACTB
|
42 |
Sigma |
1:50,000 |
Ventricle, atrium, liver, kidney, spleen, cerebrum, cerebellum, aorta, psoas muscle, femoral muscle |
Table 2.Average signal intensities in eight rats for comparison of protein expression in each gene across tissue types
|
HPRT1
|
PPIA
|
GYS1
|
TBP
|
YWHAZ
|
GAPDH
|
ACTB
|
Cerebrum |
9.82 ± 0.75 |
10.23 ± 0.95 |
9.07 ± 1.84 |
10.49 ± 1.41 |
11.27 ± 1.10 |
11.58 ± 0.56 |
9.36 ± 1.12 |
Cerebellum |
9.85 ± 0.81 |
10.92 ± 0.31 |
9.57 ± 1.84 |
9.79 ± 1.96 |
11.18 ± 0.84 |
9.17 ± 1.06 |
9.35 ± 1.05 |
Ventricle |
9.77 ± 0.89 |
9.92 ± 0.99 |
10.05 ± 1.97 |
5.63 ± 1.72 |
10.17 ± 1.92 |
11.40 ± 0.96 |
6.77 ± 1.60 |
Atrium |
9.72 ± 1.72 |
10.91 ± 1.10 |
9.83 ± 1.87 |
4.65 ± 2.10 |
10.14 ± 2.28 |
9.35 ± 0.98 |
8.10 ± 0.93 |
Psoas muscle |
9.81 ± 0.99 |
9.33 ± 2.38 |
10.10 ± 2.08 |
9.71 ± 1.69 |
7.15 ± 2.55 |
11.57 ± 1.11 |
5.79 ± 1.95 |
Femoral muscle |
10.04 ± 0.94 |
9.85 ± 1.90 |
10.01 ± 1.05 |
9.39 ± 1.44 |
6.12 ± 3.54 |
11.55 ± 1.62 |
7.50 ± 4.33 |
Liver |
9.03 ± 1.22 |
10.43 ± 1.28 |
1.99 ± 0.54 |
4.91 ± 1.08 |
9.51 ± 1.53 |
11.2 ± 1.92 |
9.51 ± 1.14 |
Spleen |
9.90 ± 0.61 |
10.41 ± 0.92 |
2.87 ± 0.72 |
9.50 ± 2.42 |
9.08 ± 0.98 |
10.60 ± 1.07 |
10.33 ± 2.29 |
Kidney |
9.57 ± 0.69 |
10.37 ± 0.53 |
1.88 ± 0.76 |
3.11 ± 1.21 |
9.77 ± 0.87 |
10.41 ± 0.73 |
7.73 ± 0.81 |
Aorta |
10.99 ± 3.17 |
9.00 ± 2.57 |
7.33 ± 5.38 |
6.41 ± 2.19 |
5.61 ± 3.44 |
9.60 ± 9.08 |
10.63 ± 2.01 |
Table 3.Coefficient of variation values (%) of each antibody according to tissue types
|
HPRT1
|
PPIA
|
GYS1
|
TBP
|
YWHAZ
|
GAPDH
|
ACTB
|
Cerebrum |
7.64 |
9.29 |
20.29 |
13.44 |
9.76 |
4.84 |
11.97 |
Cerebellum |
8.22 |
2.84 |
19.23 |
20.02 |
7.51 |
11.56 |
11.23 |
Ventricle |
9.11 |
9.98 |
19.60 |
30.55 |
18.88 |
8.42 |
23.63 |
Atrium |
17.70 |
10.08 |
19.02 |
45.16 |
22.49 |
10.48 |
11.48 |
Psoas muscle |
10.09 |
25.51 |
20.59 |
17.40 |
35.66 |
9.59 |
33.68 |
Femoral muscle |
9.36 |
19.29 |
10.49 |
15.34 |
57.84 |
14.03 |
57.73 |
Liver |
13.51 |
12.27 |
27.14 |
22.00 |
16.09 |
17.14 |
11.99 |
Spleen |
6.16 |
8.84 |
25.09 |
25.47 |
10.79 |
10.09 |
22.17 |
Kidney |
7.21 |
5.11 |
40.43 |
38.91 |
8.90 |
7.01 |
10.48 |
Aorta |
28.84 |
28.56 |
73.40 |
34.17 |
61.32 |
94.58 |
18.91 |