DEG Discovery Custom Service

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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: [email protected]

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

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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

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GeneFishingTM DEG pre-screening



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

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GeneFishingTM DEG full-screening



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

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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

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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

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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

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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

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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

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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

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1 개0 개

GP 1151 2

GP 1161 2

GP 1171 2

GP 1181 2

GP 1191 2

GP 1201 2

500bp

1000bp

67

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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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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.

DEG Discovery Custom Service

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