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PrimerArray® Hepatic Differentiation (Human)

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1. SYBR Molecular Probe Inc http wwwr takara bio coJp 15 PH017 Iaf4R4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJjp v201307Da
2. PrimerArray 5 PCR PCR PCR PCR 1 PCR 2 PCR 1 3 PCR Hold Cycle 1 95 30 2 Step PCR Cycle 40 95 C 5 te mn 60 30 mm mm _ Dissociation http www takara bio coJp 9 PH017 6 PCR PCR PrimerArray Analysis Tool for Hepatic Differentiation Multiple RQ 1
3. 2 PrimerArray PCR 4 ul PCR OOOOO PrimerArray Test Sample http www takara bio coJp 8 PH017 3 PCR Sealing Film Plate Sealing Pads 9090 Sealing Film PCR Sealing Film PCR PCR 4 24 4 PrimerArray
4. PrimerArray 4 EE 1 PrimerArray 20 A NS i PrimerArray KC OO PrimerArray 4 0 2 RNA RNA IV Appendix 1 RNA 2 1 RNA NucleoSpin RNA 740955 10 50 250
5. IV Appendix 2 PCR 4 PCR 1 2 PCR 4 1 3 cDNA SYBR Premix Ex Tag Ti RNaseH Plus RR820A PCR Control Sample Test Sample 1 cDNA cDNA PCR 1 110 SYBR Premix Ex Tag 1 Tli RNaseH Plus 2 Xx 12 5 ul 1 375 ul cDNA 50 ng ul 0 4 ul 44 ul dH2O 8 1 ul 891 ul Total 21 ul 2 310 ul total RNA http www takara bio coJp 7
6. RNA NucleoSpin RNA 740955 10 50 250 RNAiso Plus 9108 9109 DNase Recombinant DNase RNase free 2270A PrimeScript RT Master Mix Perfect Real Time RR036A PCR SYBR Premix Ex Tag Il Tli RNaseH Plus RR820A PrimerArray Analysis Tool for Hepatic Differentiation Multiplate RQ http www takara bio coJp 4 PH017 20 1 NH A 4 DD GR RG PrimerArray 20 PrimerArray
7. Perfect Real Time http wwwr takara bio coJp realtime SYBR Green RT PCR 5 07 2 RefSed 2005 3 4 PCR 120 126 2008 PCR 3 1 RT PCR 39 43 Cai J et al 2007 Hepatology 45 5 1229 1239 Kazutoshi Takahashi et al 2007 Cell 131 861 872 Ann DeLaForest et al 2011 Development 138 4143 4153 Kazuo Takayama et al 2012 Journal of Hepatology Vol 57 628 636 Yue Yu etal 2012 Stem Cell Research 9 196 207
8. 1 2 Thermal Cycler Dice Real Time System MRO TP960 Thermal Cycler Dice Real Time System Single MRO TP870 Multiplate RQ Thermal Cycler Dice Real Time System RNA 1 PCR 2 5 3 5 cDNA 20 RNA 30 RNA DNase 1 2 5 ug total RNA 20 PrimeScript RT Master Mix Perfect Real Time RR036A total RNA cDNA
9. 2 total RNA 2 PCR 42 ul 3 2 PrimerArray 8 ul http wwwr takara bio coJp 13 PH017 4 PCR Hold Cycle 1 95 30 2 Step PCR Cycle 40 95 5 60C 31or34 3 Dissociation 3 7300 31 7500 34 Applied Biosystems 7500 Fast Real Time PCR System Life Technologies 1 cDNA PCR 1 110 SYBR Premix Ex Tag ll Ti RNaseH Plus 2 Xx 10 ul 1 100 ul ROX Reference Dye ll 50X 0 4 wl 44 wl cDNA 50 ng ul 0 4 ul 44 ul dH2O 6 ul 660 ul Total 16 8 ul 1 848 wl total RNA 2 PCR 16 8 ul 3 2 PrimerArray 3 2 ul 4 PCR Hold
10. PCR 10 cDNA SYBR Premix Ex 7gg 1 Tli RNaseH Plus RR820A PCR Control Sample Test Sample http wwwr takara bio coJp 2 PH017 5 8 PrimerArray PCR Control Sample PrimerArray PCR 1 2 PCR PCR Control Sample Test Sample Win ti FE te fe Ciera ee H FE ER SL ET NT SO Er a rr a ta raw M 30 Ct A A Ct 1E 02 1E 01 1E 00 _ 1 E 1E O1 8 mt 1E 02 i tka UB tr Eo 10n 1E 04 LE 1 1E 05 1 a EF 1E 05 1E 04 1E 03 1E 02 1E 01 1E 00 1E 01 1E 02 Control Sample
11. http www takara bio coJp 3 PH017 PrimerArray Hepatic Differentiation Human 50 lx 88 50 ulx 8 Forward amp Reverse primer mix 2 5 uM 25 ul 12 CR 0 Excel http www takara Dbio CO jD Drimerarray_tool3 Mm rr mn PCR Thermal Cycler Dice System TP900 TP960 PCR 8
12. PrimerArray Analysis Tool for Hepatic Differentiation PrimerArray Analysis Tool for Hepatic Differentiation 1 Scatter Plot Fold Difference 3D Profle Scatter Plot 3D Profile 1 Fold Difference Test Sample http www takara bio co jp TE 05 1E 05 1E 04 1E 03 1E 02 Control Sample 1E 01 1E 00 1E 01 1E 02 Scatter Plot 1 n 1 1o0 00 3D Profile Fold Change 11 PHO17 Multiplate RQ Relative Quantity Chart Gene Profile Relative Quantity Chart 1M mlre ia FE F Cam akira EEELLaiH UEFHa Gene Profle 1
13. Recombinant DNase RNase free 2270A DNA DNase 1 total RNA X ul 20 50 ug 10 x DNase Buffer 5 ul 1 x RNase Inhibitor 40 U ul 0 5 ul 20 U Recombinant DNase RNase free 2 ul 10U DEPC 425 ul 50 up 2 37 20 3 DNase A 1 2 5 ul 0 5 M EDTA 80 2 2 DEPC 100 fup B 1 50 ul DEPC 100 ul 25 24 1 2 15 000 rpm 5 3 24 1 4 15 000 rpm 5 4 10 ul 3 M 250 ul
14. PHO1 IdRaRa PrimerArray PrimerArray Hepatic Differentiation Human v201307Da PrimerArray Hepatic Differentiation Human PCR 1 Array RT PCR PrimerArray AnalysisTool for Hepatic Differentiation 1 Multiplate RQ 2 1 URL http www takara Dbio CoJp Drimerarray_tool3
15. PH017 2 PCR 21 ul 8 1 8 13 273 ul 2 8 PCR 21 ul Y 9 YOOOOO oo 6 0222882222 3282 9238882 XOXOXOXXXXO OOOOCKOCXOOCXO 1838 39323 O QOOOOOOOC i Control Sample Test Sample 4 2 PCR PrimerArray 1 PrimerArray PrimerArray PrimerArray
16. Cycle 1 95 C 30 2 Step PCR Cycle 40 95 3 60 30 Dissociation http wwwr takara bio coJp 14 PH017 VII PrimeScript RT Master Mix Perfect Real Time RR036A B SYBR Premix Ex Tag 11 Tli RNaseH Plus RR820S A B L W PCR Thermal Cycler Dice Real Time System TP900 Thermal Cycler Dice Real Time System MRO TP960 Thermal Cycler Dice Real Time System Single TP850 Thermal Cycler Dice Real Time System Single MRO TP870 96well Hi Plate for Real Time NJ400 Sealing Film for Real Time NJ500 NucleoSpin RNA 740955 10 50 250 RNAiso Plus 9108 9109 Recombinant DNasel RNase free 2270A B PrimerArray Primer Set ID 5
17. PrimerArray Analysis Tool for Hepatic Differentiation Ct Excel CSV Multiplate RQ Gt Multiplate RQ Thermal Cycler Dice Real Time System Multiplate RQ PCR Omit PrimerArray Analysis Tool for Hepatic Differentiation http wwwr takara bio coJp 10 PH017 4
18. PCR a LT P LT 1 3 5 i _ m i TL ia i Threshold PCR Log scale Threshold 2 Ct Tm Ct Tm PCR 3
19. RNAiso Plus 9108 9109 RNA total RNA RNA DNA TE Buffer 250 ng u 2 2 DNA total RNA DNA DNA PCR DNA total RNA DNase DNA NucleoSpin RNA 740955 10 50 250 RNA DNase RNA DNase http www takara bio co jp 5 PH017 DNase DNA total RNA
20. 1 ua Fr IF se En hl 1 13 5 FR HERE http wwwr takara bio coJp 12 PH017 IV Appendix 1 RNA RNA RNase RNase RNA RNase RNA 1 2 1 180 60 2 0 1 DEPC 37 1 DEPC 120 30 RN
21. gt 99 10 5 4 15 000 rpm 15 6 70 4 15 000 rpm 5 7 8 DEPC http www takara bio coJp 6 PH017 3 2 RNA total RNA PrimeScript RT Master Mix Perfect Real Time RR036A 1 1 5 Xx PrimeScript RT Master Mix 10 ul 1 Xx total RNA 250 ng ul 10 ul 2 5 ud RNase Free dH2O 30 ul Total 50 ul 2 37 15 85 5 4 4 6 PCR Thermal Cycler Dice Real Time System
22. A RNA 80 60 DEPC 0 1 DEPC RNA 2 PCR I Applied Biosystems 7300 7500 Real Time PCR System Life Technologies 1 cDNA PCR 1 110 SYBR Premix Ex Tag ll Ti RNaseH Plus 2 Xx 25 ul 2 750 ul ROX Reference Dye or Dye 11 50 xX 1 1 Al 110 ul cDNA 50 ng ul 2 0 8 ul 88 ul dH2O 15 2 ul 1 672 ul Total 42 ul 4 620 ul 1 Applied Biosystems 7500 Real Time PCR System ROX Reference Dye ll 7300 Real Time PCR System ROX Reference Dye

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