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DEG Discovery Custom Service

Total RNA Electrophoresis. 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) GeneFishing TM DEG pre- and full-screening.

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DEG Discovery Custom Service

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  1. Total RNA Electrophoresis 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 • OD determination • Electrophoresis of 3 ug total RNA 1 2 www.seegene.com

  2. GeneFishingTM DEG pre-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com 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 1 GP 9 1 2 GP 10 1 2 GP 11 1 2 GP 12 1 2 GP 13 1 2 GP 14 1 2 GP 8 1 2 3 2 www.seegene.com

  3. GeneFishingTM DEG pre-screening 500bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 16 1 2 GP 17 1 2 GP 18 1 2 GP 19 1 2 GP 20 1 2 GP 15 1 2 5 7 8 6 4 www.seegene.com

  4. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 22 1 2 GP 23 1 2 GP 24 1 2 GP 25 1 2 GP 26 1 2 GP 27 1 2 GP 21 1 2 10 9 11 GP 29 1 2 GP 30 1 2 GP 31 1 2 GP 32 1 2 GP 33 1 2 GP 34 1 2 GP 28 1 2 14 16 12 13 15 www.seegene.com

  5. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 36 1 2 GP 37 1 2 GP 38 1 2 GP 39 1 2 GP 40 1 2 GP 35 1 2 18 20 17 19 21 22 GP 42 1 2 GP 43 1 2 GP 44 1 2 GP 45 1 2 GP 46 1 2 GP 47 1 2 GP 41 1 2 23 26 25 24 www.seegene.com

  6. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 49 1 2 GP 50 1 2 GP 51 1 2 GP 52 1 2 GP 53 1 2 GP 54 1 2 GP 48 1 2 31 29 32 30 33 27 34 28 GP 56 1 2 GP 57 1 2 GP 58 1 2 GP 59 1 2 GP 60 1 2 GP 55 1 2 35 36 www.seegene.com

  7. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 62 1 2 GP 63 1 2 GP 64 1 2 GP 65 1 2 GP 66 1 2 GP 67 1 2 GP 61 1 2 GP 69 1 2 GP 70 1 2 GP 71 1 2 GP 72 1 2 GP 73 1 2 GP 74 1 2 GP 68 1 2 39 40 38 41 42 37 www.seegene.com

  8. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 76 1 2 GP 77 1 2 GP 78 1 2 GP 79 1 2 GP 80 1 2 GP 75 1 2 44 45 43 GP 82 1 2 GP 83 1 2 GP 84 1 2 GP 85 1 2 GP 86 1 2 GP 87 1 2 GP 81 1 2 47 48 49 46 50 www.seegene.com

  9. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 89 1 2 GP 90 1 2 GP 91 1 2 GP 92 1 2 GP 93 1 2 GP 94 1 2 GP 88 1 2 56 51 55 52 54 53 GP 96 1 2 GP 97 1 2 GP 98 1 2 GP 99 1 2 GP 100 1 2 GP 95 1 2 59 57 60 58 www.seegene.com

  10. GeneFishingTM DEG full-screening 500bp 500bp 1000bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 102 1 2 GP 103 1 2 GP 104 1 2 GP 105 1 2 GP 106 1 2 GP 107 1 2 GP 101 1 2 62 63 65 61 64 GP 109 1 2 GP 110 1 2 GP 111 1 2 GP 112 1 2 GP 113 1 2 GP 114 1 2 GP 108 1 2 66 www.seegene.com

  11. GeneFishingTM DEG full-screening 500bp 1000bp DEG Discovery Custom Service Seegene Institute of Life Science, 142-21 Samsung-dong, Gangnam-gu, Seoul, 135-090, Korea E-mail: service@seegene.com GP 116 1 2 GP 117 1 2 GP 118 1 2 GP 119 1 2 GP 120 1 2 GP 115 1 2 67 www.seegene.com

  12. 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 ultra-purified 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. www.seegene.com

  13. 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. www.seegene.com

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