DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
4. Experiments
4.1 DEG Discovery Screening
(1) GeneFishingTM DEG pre- and full-screening
Total RNA Electrophoresis
1
2
• OD determination
• Electrophoresis of 3 ug total RNA
Sample
A260
A260/A280
Conc.
(ug/ul)
Total
(ug)
1. 유모
0.079
1.386
0.316
47.40
2. 무모
0.057
1.326
0.228
34.20
1
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG pre-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
3개
0개
GP 1
GP 2
GP 3
GP 4
GP 5
GP 6
GP 7
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1000bp
1
500bp
GP 8
GP 9
GP 10
GP 11
GP 12
GP 13
GP 14
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1000bp
500bp
2
3
2
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG pre-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
3개
2개
GP 15
GP 16
GP 17
GP 18
GP 19
GP 20
1
1
1
1
1
1
2
2
2
2
2
2
1000bp
500bp
5
6
7
8
4
3
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 21
1
2
GP 22
1
2
GP 23
1
2
GP 24
1
2
4개
4개
GP 25
1
2
GP 26
1
2
GP 27
1
2
GP 33
1
2
GP 34
1
2
10
1000bp
500bp
9
11
GP 28
1
2
GP 29
1
2
GP 30
1
2
GP 31
1
2
GP 32
1
2
1000bp
14
500bp
12
13
16
15
4
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 36
1
2
GP 35
1
2
GP 37
1
2
1000bp
500bp
GP 38
1
2
GP 39
1
2
GP 40
1
2
18
17
20
19
21
GP 42
1
2
GP 41
1
2
1000bp
5개
5개
GP 43
1
2
GP 44
1
2
22
GP 45
1
2
GP 46
1
2
GP 47
1
2
23
500bp
25
26
24
5
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 49
1
2
GP 48
1
2
GP 50
1
2
GP 51
1
2
GP 52
1
2
8개
2개
GP 53
1
2
GP 54
1
2
31
1000bp
29
32
30
500bp
33
27
34
28
GP 55
1
2
GP 56
1
2
GP 57
1
2
GP 58
1
2
GP 59
1
2
GP 60
1
2
1000bp
35
36
500bp
6
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 62
1
2
GP 61
1
2
GP 63
1
2
GP 64
1
2
1개
5개
GP 65
1
2
GP 66
1
2
GP 67
1
2
GP 72
1
2
GP 73
1
2
GP 74
1
2
1000bp
500bp
GP 69
1
2
GP 68
1
2
GP 70
1
2
GP 71
1
2
1000bp
39
500bp
38
40
41
42
37
7
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 75
1
2
GP 76
1
2
GP 77
1
2
GP 78
1
2
1000bp
4개
4개
GP 79
1
2
GP 80
1
2
44
45
43
500bp
GP 81
1
2
GP 82
1
2
GP 83
1
2
GP 84
1
2
GP 85
1
2
GP 86
1
2
GP 87
1
2
1000bp
47
48
500bp
46
49
50
8
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 89
1
2
GP 88
1
2
GP 90
1
2
GP 91
1
2
GP 92
1
2
6개
4개
GP 93
1
2
1000bp
GP 94
1
2
56
51
500bp
55
52
53
GP 95
1
2
GP 96
1
2
GP 97
1
2
54
GP 98
1
2
GP 99
1
2
GP 100
1
2
1000bp
59
500bp
57
58
60
9
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 101
1
2
GP 102
1
2
GP 103
1
2
GP 104
1
2
5개
1개
GP 105
1
2
GP 106
1
2
GP 107
1
2
1000bp
62
500bp
63
65
61
64
GP 108
1
2
GP 109
1
2
GP 110
1
2
GP 111
1
2
GP 112
1
2
GP 113
1
2
GP 114
1
2
1000bp
500bp
66
10
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
GeneFishingTM DEG full-screening
1. 유모 에서 발현량이 높은 유전자 표시 :
2. 무모 에서 발현량이 높은 유전자 표시 :
GP 115
1
2
GP 116
1
2
GP 117
1
2
GP 118
1
2
GP 119
1
2
1개
0개
GP 120
1
2
1000bp
500bp
67
11
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
5. Materials and Methods
First-strand cDNA Synthesis
Total RNAs extracted from your samples were used for the synthesis of first-strand cDNAs by reverse
transcriptase. Reverse transcription was performed for 1.5 h at 42ºC in a final reaction volume of 20 ㎕
containing 3 ㎍ of the purified total RNA, 4 ㎕ of 5  reaction buffer (Promega, Madison, WI, USA), 5 ㎕
of dNTPs (each 2 mM), 2 ㎕ of 10 μM dT-ACP1 (5’-CGTGAATGCTGCGACTACGATIIIIIT(18)-3’), 0.5 ㎕
of RNasin RNase Inhibitor (40 U/ ㎕; Promega), and 1 ㎕ of Moloney murine leukemia virus reverse
transcriptase (200 U/ ㎕; Promega). First-strand cDNAs were diluted by the addition of 80 ㎕ of ultrapurified water for the GeneFishingTM PCR, and stored at -20ºC until use.
ACP-based GeneFishingTM PCR
Differentially expressed genes were screened by ACP-based PCR method (Kim et al., 2004) using the
GeneFishingTM DEG kits (Seegene, Seoul, South Korea). Briefly, second-strand cDNA synthesis was
conducted at 50ºC during one cycle of first-stage PCR in a final reaction volume of 20 ㎕ containing 3-5
㎕ (about 50 ng) of diluted first-strand cDNA, 1 ㎕ of dT-ACP2 (10 μM), 1 ㎕ of 10 μM arbitrary ACP, and
10 ㎕ of 2  Master Mix (Seegene). The PCR protocol for second-strand synthesis was one cycle at
94ºC for 1 min, followed by 50ºC for 3 min, and 72ºC for 1 min. After second-strand DNA synthesis was
completed, the second-stage PCR amplification protocol was 40 cycles of 94ºC for 40 s, followed by
65ºC for 40 s, 72ºC for 40 s, followed by a 5 min final extension at 72ºC. The amplified PCR products
were separated in 2% agarose gel stained with ethidium bromide.
12
www.seegene.com
DEG Discovery Custom Service
Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea
E-mail: service@seegene.com
6. References
Hwang, I. T., Y. J. Kim, S. H. Kim, C. I. Kwak, Y. Y. Gu, and J. Y. Chun. 2003. Annealing control
primer system for improving specificity of PCR amplification. BioTechniques 35:1180-1184.
Hwang, K. C., X. S. Cui, S. P. Park, M. R, Shin, S. Y. Park, E. Y. Kim, and N. H. Kim. 2004.
Identification of differentially regulated genes in bovine blastocysts using an annealing control primer
system. Mol. Reprod. Dev. 69:43-51.
Hwang, K. C., H. Y. Lee, S. S. Cui, J. H. Kim, and N. H. Kim. 2005. Identification of Maternal mRNAs
in porcine parthenotes at the 2-cell stage: a comparison with the blastocyst stage. Mol. Reprod. Dev.
70:314-323.
Kim, Y. J., C. I. Kwak, Y. Y. Gu, I. T. Hwang, and J. Y. Chun. 2004. Annealing control primer system
for identification of differentially expressed genes on agarose gels. BioTechniques 36:424-426, 428,
430.
Kottom, T. J., and A. H. Limper. 2004. Pneumocystis carinii cell wall biosynthesis kinase gene CBK1
is an environmentally responsive gene that complements cell wall defects of cbk-deficient yeast.
Infect. Immun. 72:4628-4636.
Lee, A. Y., N. H. Kim, and S. W. Park. 2004. All trans-retinoic acid (ATRA) elevated eukaryoic
translation initiation factor 4A1 (eIF4A1) mRNA in ATRA-responsive vitiliginous epidermis. Pigment
Cell RES. 17:659-667.
Pohjanvirta, R. 2004. Comparison of several hot-start Tag DNA polymerases for detection of
differentially expressed genes by GeneFishing. Biochemica 2:17-18.
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