1 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea 4. Experiments 4.1 DEG Discovery Screening (1) GeneFishing TM DEG pre- and full-screening Total RNA Electrophoresis 1 2 SampleA260A260/A280 Conc. (ug/ul) Total (ug) 1. 유모 무모 OD determination Electrophoresis of 3 ug total RNA

2 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG pre-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 3 개 0 개 GP GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp 1 2 3

3 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG pre-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 3 개 2 개 GP GP GP GP GP GP bp 1000bp

4 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 4 개 GP GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp

5 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 5 개 GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp

6 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 8 개 2 개 GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp

7 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 1 개 5 개 GP GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp

8 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 4 개 GP GP GP GP GP GP bp 1000bp 43 GP GP GP GP GP GP GP bp 1000bp

9 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 6 개 4 개 GP GP GP GP GP GP bp 1000bp GP GP GP GP GP GP GP bp 1000bp

10 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 5 개 1 개 GP GP GP GP GP GP GP GP GP GP GP GP GP GP bp 1000bp 500bp 1000bp

11 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea GeneFishing TM DEG full-screening 1. 유모 에서 발현량이 높은 유전자 표시 : 2. 무모 에서 발현량이 높은 유전자 표시 : 1 개 0 개 GP GP GP GP GP GP bp 1000bp 67

12 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea 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 GeneFishing TM PCR, and stored at -20ºC until use. ACP-based GeneFishing TM PCR Differentially expressed genes were screened by ACP-based PCR method (Kim et al., 2004) using the GeneFishing TM 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.

13 DEG Discovery Custom Service Seegene Institute of Life Science, Samsung-dong, Gangnam-gu, Seoul, , Korea 6. References Hwang, I. T., Y. J. Kim, S. H. Kim, C. I. Kwak, Y. Y. Gu, and J. Y. Chun Annealing control primer system for improving specificity of PCR amplification. BioTechniques 35: Hwang, K. C., X. S. Cui, S. P. Park, M. R, Shin, S. Y. Park, E. Y. Kim, and N. H. Kim Identification of differentially regulated genes in bovine blastocysts using an annealing control primer system. Mol. Reprod. Dev. 69: Hwang, K. C., H. Y. Lee, S. S. Cui, J. H. Kim, and N. H. Kim Identification of Maternal mRNAs in porcine parthenotes at the 2-cell stage: a comparison with the blastocyst stage. Mol. Reprod. Dev. 70: Kim, Y. J., C. I. Kwak, Y. Y. Gu, I. T. Hwang, and J. Y. Chun Annealing control primer system for identification of differentially expressed genes on agarose gels. BioTechniques 36: , 428, 430. Kottom, T. J., and A. H. Limper 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: Lee, A. Y., N. H. Kim, and S. W. Park All trans-retinoic acid (ATRA) elevated eukaryoic translation initiation factor 4A1 (eIF4A1) mRNA in ATRA-responsive vitiliginous epidermis. Pigment Cell RES. 17: Pohjanvirta, R Comparison of several hot-start Tag DNA polymerases for detection of differentially expressed genes by GeneFishing. Biochemica 2:17-18.