Post on 02-Jan-2016
description
1
DEG Discovery Custom Service
www.seegene.com
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
Sample A260 A260/A280Conc.
(ug/ul)
Total
(ug)
1. 유모 0.079 1.386 0.316 47.40
2. 무모 0.057 1.326 0.228 34.20
• OD determination
• Electrophoresis of 3 ug total RNA
2
DEG Discovery Custom Service
www.seegene.com
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 :
3 개 0 개
GP 2
1 2
GP 3
1 2
GP 4
1 2
GP 5
1 2
GP 6
1 2
GP 7
1 2
GP 1
1 2
GP 8
1 2
GP 9
1 2
GP 10
1 2
GP 11
1 2
GP 12
1 2
GP 13
1 2
GP 14
1 2
500bp
1000bp
500bp
1000bp
1
2 3
3
DEG Discovery Custom Service
www.seegene.com
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 :
3 개2 개
GP 15
1 2
GP 16
1 2
GP 17
1 2
GP 18
1 2
GP 19
1 2
GP 20
1 2
500bp
1000bp
4
56 7 8
4
DEG Discovery Custom Service
www.seegene.com
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 :
4 개4 개
GP 211 2
GP 221 2
GP 231 2
GP 241 2
GP 251 2
GP 261 2
GP 271 2
GP 281 2
GP 291 2
GP 301 2
GP 311 2
GP 321 2
GP 331 2
GP 341 2
500bp
1000bp
500bp
1000bp
9
10
11
1213
14
15
16
5
DEG Discovery Custom Service
www.seegene.com
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 :
5 개5 개
GP 351 2
GP 361 2
GP 371 2
GP 381 2
GP 391 2
GP 401 2
GP 411 2
GP 421 2
GP 431 2
GP 441 2
GP 451 2
GP 461 2
GP 471 2
500bp
1000bp
500bp
1000bp
17
18
19 20
21 22
23
24
25 26
6
DEG Discovery Custom Service
www.seegene.com
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 :
8 개2 개
GP 481 2
GP 491 2
GP 501 2
GP 511 2
GP 521 2
GP 531 2
GP 541 2
GP 551 2
GP 561 2
GP 571 2
GP 581 2
GP 591 2
GP 601 2
500bp
1000bp
500bp
1000bp
27
28
29
30
31
32
3334
35 36
7
DEG Discovery Custom Service
www.seegene.com
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 :
1 개5 개
GP 611 2
GP 621 2
GP 631 2
GP 641 2
GP 651 2
GP 661 2
GP 671 2
GP 681 2
GP 691 2
GP 701 2
GP 711 2
GP 721 2
GP 731 2
GP 741 2
500bp
1000bp
500bp
1000bp
37
38
39
4041 42
8
DEG Discovery Custom Service
www.seegene.com
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 :
4 개4 개
GP 751 2
GP 761 2
GP 771 2
GP 781 2
GP 791 2
GP 801 2
500bp
1000bp
43
GP 811 2
GP 821 2
GP 831 2
GP 841 2
GP 851 2
GP 861 2
GP 871 2
500bp
1000bp
4445
46
47 48 49
50
9
DEG Discovery Custom Service
www.seegene.com
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 :
6 개4 개
GP 951 2
GP 961 2
GP 971 2
GP 981 2
GP 991 2
GP 1001 2
500bp
1000bp
GP 881 2
GP 891 2
GP 901 2
GP 911 2
GP 921 2
GP 931 2
GP 941 2
500bp
1000bp
51
52
53
54
55
56
57
58
59
60
10
DEG Discovery Custom Service
www.seegene.com
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 :
5 개1 개
GP 1011 2
GP 1021 2
GP 1031 2
GP 1041 2
GP 1051 2
GP 1061 2
GP 1071 2
GP 1081 2
GP 1091 2
GP 1101 2
GP 1111 2
GP 1121 2
GP 1131 2
GP 1141 2
500bp
1000bp
500bp
1000bp
61
62
63
64
65
66
11
DEG Discovery Custom Service
www.seegene.com
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 :
1 개0 개
GP 1151 2
GP 1161 2
GP 1171 2
GP 1181 2
GP 1191 2
GP 1201 2
500bp
1000bp
67
12
DEG Discovery Custom Service
www.seegene.com
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 SynthesisTotal 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 ultra-purified water for the GeneFishingTM PCR, and stored at -20ºC until use.
ACP-based GeneFishingTM PCRDifferentially 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.
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DEG Discovery Custom Service
www.seegene.com
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.