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1. siRNA W ST 8 ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 007 GenomONE Si Technical Data Sheet SIRNA transfection Negative control siRNA 2 CDC2 siRNA 1 8 1 6 gt Sw 1 4 2 1 2 3 o gt N 0 8 E 0 6 g 0 4 0 2 0 Control PBS Product X ProductR GenomONE si Relative Quantity qRT PCR assays n 3 Raji CDC2 siRNA CDC2 siRNA 50nM transfection gt 2days gt RT PCR mRNA 27277 Z 97 1 Cell Raji Human Burkitt lymphoma cell line ATCC CCL 86 Culture condition 1 25 x 105cells well 500uL 10 FBS RPM 1640 Culture plate 24 well plate FALCON 3047 siRNA Very High Potency Hs_CDC2 siRNA QIAGEN Cat No 1027273 Negative control 1 siRNA Ambion Code No AM4639 Step 24 well plate HVJ E HVJ E 2 5uL D D SIRNA 10u
2. SK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 003 GenomONE Si Technical Data Sheet Jurkat SIRNA transfection Negative control siRNA ml Eg5 siRNA Oo 73 Absorbance 450nm Oo NO Control PBS ProductX ProductR GenomONE si Jurkat Eg5 siRNA Eg5 siRNA 50nM transfection gt 2days WST 8 A_ 27277 Z 97 1 Cell Jurkat clone E6 1 Human T cell leukemia cell line ATCC TIB 152 Culture condition 2 x 104cells well 100uL 10 FBS RPMI 1640 Culture plate 96 well plate IWAKI 3860 096 siRNA Silencer KIF11 Eg5 siRNA Ambion Code No AM4639 Negative control 1 siRNA Ambion Code No AM4639 HVJ E D D siRNA 10uM E HVJ E well 37C 5 CO 48
3. LPS Cyclophilin B PPIB peptidylprolyl isomerase B 1 Western blotting ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 003 GenomONE Si Technical Data Sheet BALB c mouse primary B cells miRNA transfection Cyclophilin B B actin a ell Viabili a b c d e f g h y 9 rr 100 Day 1 a 1 y y 80 GN Day 2 Day 2 0 NN 40 Control PBS vV f Mock 9 Day 3 mW Day 3 a e Control PBS Cyclophilin B miRNA b f Mock 0 c g Negative control miRNA 50nM d h Cyclophilin B miRNA 50nM 20 Negative control miRNA 0 1 2 3 Day Cell Viability Pl staining assay Mouse primary B cell Cyclophilin B miRNA Post stimulation Stimulation B cells isolation amp Transfection Western blotting 1 3 days GenomO0NE i 1 Cell Mouse primary B cells isolated from spleen of female BALB c mouse 12 weeks old Stimulation LPS 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640
4. a i siRNA 5 F HN enomd0NME f siRNA References Review articles Kaneda Y et al Hemagglutinating virus of Japan HVJ envelope vectorasa versatile gene delivery system Molecular Therapy 6 219 226 2002 Kaneda Y New vector innovation for drug delivery development of fusigenic non viral particles Curr Drug Targets 4 8 599 602 2003 Kaneda Y Applications of Hemagglutinating Virus of Japan in therapeutic delivery systems Expert Opin Drug Deliv 5 2 221 233 2008 Zhang Q etal HVJ envelope vector a versatile delivery system its development application and perspectives Biochem Biophys Res Commun 373 345 349 2008 Kato F et al Hemagglutinating Virus of Japan Envelope Vectors as High Performance Vehicles for Delivery of Small RNAs J Genet Syndr Gene Ther in press 2013 GenomONE Si Technical Data Sheet U937 Raji THP 1 Jurkat HL 60 siRNA GenomONE Si Technical Data Sheet U937 SIRNA transfection Negative control siRNA 0 9 Eg5 siRNA 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 9476 Absorbance 450nm Control PBS ProductX ProductR GenomONE si U937 Eg5 siRNA Eg5 siRNA 50nM transfection gt 2days WST 8 A_ g 777Z 97
5. 1 Cell U 937 Human leukemic monocytic lymphoma cell line ATCC CRL 1593 2 Culture condition 4 x 103cells well 100uL 10 FBS RPMI 1640 Culture plate 96 well plate IWAKI 3860 096 siRNA Silencer KIF11 Eg5 siRNA Ambion Code No AM4639 Negative control 1 siRNA Ambion Code No AM4639 96 well plate HVJ E HVJ E 2 5uL D lt D SiRNA 10uM E HVJ E well 37C 5 CO 48 HVJ E 0 9uL well ZZg 7 77 7 9 U937 Eg5 siRNA 94 2g 27777 9 Eg5 KIF 11
6. 1 Real Time PCR W estern blotting HVJ E 4 5uL well SK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 006 GenomONE Si Technical Data Sheet BALB c mouse primary T cells miRNA transfection Negative Control miRNA Cyclophilin B miRNA Control PBS Product X Product R GenomONE si NA o NM Relative Quantity OO normalized 18Sr Relative Quantity qRT PCR assays n 3 Pre stimulated mouse primary T cells Cyclophilin B miRNA Stimulation T cells isolation amp No stimulation RT PCR mRNA 1 day Transfection 2 days Splenocyte 2 777 97 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 9 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX WW 100 x Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mi
7. HVJ E well 37C 5 CO 48 HVJ E 4 5uL well Mouse spleen cells PMA ionomycin 1 T cells g 27 Z 97 Cyclophilin B control siRNA RT PCR 70 50nM siRNA 8696 Cyclophilin B PPIB peptidylprolyl isomerase B 1 Real Time PCR SK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 006 GenomONE Si Technical Data Sheet C57BL 6 mouse primary T cells miRNA transfection sm Relative Quantity pCell Viability 1 6 90 14 80 70 60 10 a ww pe 50 lt 40 amp 0 6 61 a 20 94 85 0 2 bd 10 00 8 1 1 0 Control PBS Negative 10nM 2nM 0 4nM 0 08nM control miRNA Rela
8. lt HVJ E 0 9uL well 77 77 7 9 Jurkat Eg5 siRNA 73 2 277 Z 9 Eg5 KIF 11 siRNA W ST 8 ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 004 GenomONE Si Technical Data Sheet HL 60 SIRNA transfection 1 2 Negative control siRNA CDC2 siRNA Co Relative quantity normalized 18S rRNA I Control PBS Product X ProductR GenomONE si Relative Quantity qRT PCR assays n 3 HL 60 CDC2 siRNA CDC2 siRNA 50nM transfection gt 2day
9. 6em0m0NME 10nM Cyclophilin B control miRNA RT PCR 94 Cyclophilin B PPIB peptidylprolyl isomerase B 1 Real Time PCR ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 005 GenomONE Si Technical Data Sheet BALB c mouse primary T cells miRNA transfection a Relative Quantity a b c d e f Cell Viability 70 1 2 1 0 60 0 8 56 L 50 ti 3 1 93o6 0 og B actin 5 og 75 30 5 20 a Negative control miRNA 1000pM a 10 b Cyclophilin B miRNA 1000pM 0 0 0 Ci Cyclophilin B miRNA 400pM Control PBS 400pM 400pM J 100pM 25pM 6 3pM d Cyclophilin B miRNA 100pM prt odn e Cyclophilin B miRNA 25pM miRNA f Cyclophilin B miRNA 6 3pM Relative Quantity qRT PCR assays n 3 Cell Viability Pl staining assay Pre stimulated mouse primary T cells Cyclophilin B miRNA Stimulation T cells isolation amp No stimulation RT PCR mRNA SD 1 day Transfection 2 days Western blo
10. 48 HVJFE 4 5uUwel lt 277 7 AZ 97 LPS mouse primary B cells Mimic Housekeeping Positive Control 1 PPIB miRNA 82 4g Z77 Z 97 LPS Cyclophilin B PPIB peptidylprolyl isomerase B 1 Rea Time PCR 0120 409 816 FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 002 GenomONE Si Technical Data Sheet BALB c mouse primary B cells miRNA transfection a b C d e Cyclophilin B M000N R mmT 40 Bs actin a a Control PBS Cell Viability b Mock 50nM 50nM 10nM Negative control miRNA 50nM Control PBS Mock Negative cyclophilin B miRNA d Cyclophilin B miRNA 50nM 0 e Cyclophilin B miRNA 10nM Cell Viability Pl staining assay Pre stimulated mouse primary B
11. High Throuhput Screening HTS 2 assays wells wells le 0 ite 0 le 100 2000 28000 30240 GenomONE Si Technical Data Sheet 27777 Z 37 siRNA miRNA transfection kit HVJ E HN protein F protein EN HVJ Sendai ViruS HVJ Envelope HVJ E Hemagglutinating virus of Japan HVJ Envelope HVJ E RNA 300nm vesicle HVJ E F protein live HVJ E HVJ E SiRNA miRNA HVJ Hemagglutinating virus of Japan Sendai virus SeV Mouse Parainfluenza virus type 1 HVJ E R siRNA HVJ Envelope HVJ E HVJ E
12. GenomONE Si Technical Data Sheet BALB c mouse primary T cells siRNA transfection Ga Relative Quantity 1 2 s Cell Viabilty 70 10 w 60 50 0 8 40 lt 0 6 Co 0 4 5 67 S92 79 79 86 10 0 0 0 EontoPBS 150nM 150nM 7nM 5 6nM Negative Cyclophilin B siRNA control Relative Quantity qRT PCR assays n 3 siRNA Cell Viability Pl staining assay Pre stimulated mouse primary T cells Cyclophilin B miRNA Stimulation T cells isolation amp No stimulation RT PCR mRNA 1 day Transfection 2 days SpDlenocyte 7g 77 Z 97 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 12 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 8 x 10 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX WW 100 x Culture plate 24 well plate siRNA siGENOME Cyclophilin B control siRNA Thermo Scientific Cat No D 001136 01 05 Negative control 1 siRNA Ambion Code No AM4639 24 well plate HVJ E HVJ E 2 5uL D SiRNA 30 1 1uM E
13. BALB c mouse primary T cells SIRNA miRNA transfection siRNA transfection miRNA transfection Relative Quantity A Relative Quantity egeCell Viability Cell Viability 1 4 80 1 6 0 80 1 2 1 4 lt 60 gt 12 60 gt z 1 0 BE 5 1 0 8 0 0 8 40 gt 4 oO 0 gt 806 77 0 SEO SE 5 96 0 4 8 20 0 4 20 0 0 0 0 0 0 Control PBS Negative Control cyclophilin B Control PBS Negative Control cyclophilin B miRNA miRNA miRNA miRNA Relative Quantity qRT PCR assays n 2 3 Cell Viability Pl staining assay Pre stimulated mouse primary T cells Cyclophilin B siRNA miRNA Stimulation No stimulation Splenocyte Tcells isolation amp Transfection RT PCR mRNA 1day 2days g 77777Z 97 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 7 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX 100 x Culture plate 24 well plate siRNA siGENOME Cyclophilin B control siRNA Thermo Scientific Cat No D 001136 01 05 Negative control 1 siRNA Ambion Code No AM4639 miRNA Mimic Housekeeping Positive Contr
14. E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Ao GS S M 007 GenomONE Si Technical Data Sheet BALB c mouse primary B cells Anti CD40 LPS stimulation Anti CD40 IL4 stimulation Relative Quantity a Relative Quantity Cel Viability Cell Viability 1 4 60 1 4 50 50 12 gt 40 10 oO 10 5 021 Eo lt CO 30 lt 0 8 0 0 8 Ss 76 7 S O 5 Oo 30 0 06 RO6 7476 5 20 lt E 0 4 E 0 4 10 10 0 2 022 0 0 0 0 0 0 Control PBS 2 B Control PBS Negative contro Cyclophilin B ImiRNA ImiRNA miRNA Relative Quantity qRT PCR assays n 3 Cell Viability Pl staining assay Pre stimulated mouse primary B cells Cyclophilin B miRNA Stimulation No stimulation lsolated B cells Transfection RT PCR mRNA 1 day 2 days 7g 7777 Z 97 1 Cell Mouse primary B cells isolated from spleen of female BALB c mouse 7 weeks old Stimulation anti CD40 1ug mL LPS 1ug mL anti CD40 1ug mL IL4 34 5ng mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 10mM HEPES 50uM 2 ME C
15. Technical Data Sheet BALB c mouse primary T cells miRNA transfection cyclophilnB a b c d e Cell Viability RI 100 Product R genomohE si NR NRN Product X Product R aa OY ww U96 a B actin 10nM 10nM 2nM 0 4nM Control PBS NegatiVe cyclophilin B miRNA sg Control miRNA 277 77 6 f d h a f Control PBS Cell Viability Pl staining assay Product R b g Negative control miRNA 50nM c h Cyclophilin B miRNA 50nM d i Cyclophilin B miRNA 2nM e j Cyclophilin B miRNA 0 4nM Product X Pre stimulated mouse primary T cells Cyclophilin B miRNA Stimulation T cells isolation amp No stimulation 1 day Transfection 2 days g 777 Z 97 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 8 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX 100 x Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D Splenocyte Western blot
16. siRNA Thermo Scientific Cat No D 001136 01 05 Negative control 1 siRNA Ambion Code No AM4639 miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 HVJ E HVJ E 2 5uL D siRNA miRNA 10uM siRNAmiRNA 10uL E E 5uL HVJ E well 5 37 C 5 CO 48 HVJ E 4 5uLwel Genom0ME mouse primary B cells Mimic Housekeeping Positive Control 1 PPIB miRNA 66 Cyclophilin B control siRNA 21 Cyclophilin B PPIB peptidylprolyl isomerase B 1 Real Time PCR S 0120 409 816 FAX 06 6444 7183 E mail HVJ
17. with 10 FBS 10mM HEPES 50uM 2 ME Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 HVJ E D miRNA 10uM E HVJ E well 37C 5 CO lt HVJ E 4 5uL well 7g7277 Z 57 mouse primary B cells Mimic Housekeeping Positive Control 1 PPIB miRNA LPS 1 lt 3 Western blotting Cyclophilin B PPIB peptidylprolyl isomerase B 1 Western blotting ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 004 GenomONE Si Technical Data Sheet
18. 3 15 URL http www iskweb co jp hvj e Mo GS M 007 GenomONE Si Technical Data Sheet BALB c mouse primary B cells miRNA transfection Negative control miRNA Cyclophilin B miRNA Relative Quantity normalized 18S rRNA 82 Control PBS Product R GenomONE si Relative Quantity qRT PCR assays n 3 Pre stimulated mouse primary B cells Cyclophilin B miRNA Stimulation lsolated B cells Transfection gt RT PCR mRNA 1 day 2 days GienomONE i 1 Cell Mouse primary B cells isolated from spleen of female BALB c mouse 8 weeks old Stimulation LPS 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 10mM HEPES 50uM 2 ME Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D miRNA 10uM HVJ_E well a 37 C 5 CO
19. FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 005 GenomONE Si Technical Data Sheet Mouse primary B cells T cells siRNA miRNA GenomONE Si Technical Data Sheet BALB c mouse primary B cells SIRNA miRNA transfection miRNA transfection siRNA transfection EE Relative Quantity EE Relative Quantity Cell Viability Cell Viability 2 0 60 2 5 60 1 5 lt 3 40 wm 40 wm c 8 66 E83 5 O 0 0 ss 21 1 1 0 20 20 le Cc 5 0 5 c 0 0 0 0 0 0 Control PBS Negativecontrol CyclophilinB Control PBS Negativecontrol CydlophilinB miRNA miRNA SIRNA SIRNA Relative Quantity qRT PCR assays n 3 Cell Viability Pl staining assay Mouse primary B cells Cyclophilin B siRNA miRNA No stimulation B cells isolation amp Transfection gt RT PCR mRNA 2 days fienomOdNE i 1 Cell Mouse primary B cells isolated from spleen of female BALB c mouse 6 weeks old Culture condition 5 x 106 cells well 500uL RPMI 1640 with 10 FBS 10mM HEPES 50uM 2 ME Culture plate 24 well plate siRNA siGENOME Cyclophilin B control
20. GenomONE Si Technical Data Sheet HVJ Envelope siRNA miRNA Z77777 97 siRNA miRNA ISK 0120 409 816 FAX 06 6444 7183 2 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e 1310GS2 GenomONE Si Technical Data Sheet HVJ Envelope siRNA miRNA 277777 97 gt siRNAmiRNA 510 HTS 277777 57 HVJ Envelope HVJ E siRNA miRNA ODO 1 1 02 NHVJE SIRNA am 7 allK4NP 5
21. L wel Cyclophilin B PPIB peptidylprolyl isomerase B 1 Western blotting 0120 409 816 FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 009 GenomONE Si Technical Data Sheet Memo ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e GenomONE Si Technical Data Sheet Memo ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e
22. M E HVJ E well 37 C 5 CO 48 HVJ E 4 5uLAwel lt ZZg77 77 7 9 Raji CDC2 siRNA 75 eNomO0NE i CDC2 B M Real Time PCR ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e Mo GS S 002 GenomONE Si Technical Data Sheet HP 1 SIRNA transfection Negative control siRNA 1 4 a Eg5 siRNA 1 2 0 8 94 0 6 0 4 Absorbance 450nm 0 2 Control PBS ProductX ProductR GenomONE si THP 1 Eg5 siRNA Eg5 siRNA 50nM transfection gt 2days WST 8 A4 g 77777Z 97 1 Cell THP 1 Huma
23. cells Cyclophilin B miRNA Stimulation lsolated B cells gt Transfection Western blotting 1 day 2 days g 777 97 1 Cell Mouse primary B cells isolated from spleen of female BALB c mouse 11 weeks old Stimulation LPS 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 10mM HEPES 50uM 2 ME Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D D miRNA 10 2LM E HVJ E well F 37 C 5 CO 48 HVJ E 4 5nLAwel Genom0ANME i LPS mouse primary B cells Mimic Housekeeping Positive Control 1 PPIB miRNA 10 50nM Western blotting
24. i a e Control PBS b f Mock c g Negative control miRNA 50nM d h Cyclophilin B miRNA 50nM Mouse primary T cells Cyclophilin B miRNA Post stimulation Stimulation T cells isolati T fecti W lotti cells isolation amp Transfection 12 48 h estern blotting 2777774 97 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 12 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX W 100 x Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate E 5uL x 1 2 HVJ E well 37C 5 CO lt 7g72777 Z 7 mouse primary T cells Mimic Housekeeping Positive Control 1 PPIB miRNA PMA ionomycin 24 lt 48 Western blotting HVJ E 4 5u
25. mic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D miRNA 2uM E HVJ E well 37C 5 CO 48 HVJ E 4 5uL well Mouse spleen cells PMA ionomycin 1 T cells 2 277 Z 7 Cyclophilin B control miRNA RT PCR 79 7 Cyclophilin B PPIB peptidylprolyl isomerase B 1 Real Time PCR 0120 409 816 FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 007 GenomONE Si
26. n acute monocytic leukemia cell line ATCC TIB 202 Culture condition 2 5 x 104cells well 100uL 10 FBS RPMI 1640 Culture plate 96 well plate IWAKI 3860 096 siRNA Silencer KIF11 Eg5 siRNA Ambion Code No AM4639 Negative control 1 siRNA Ambion Code No AM4639 HVJ E D D siRNA 10uM E HVJ E well 37C 5 CO 48 lt HVJ E 0 9uL well 277777 Z 9 THP 1 Eg5 siRNA 94 27277 7 97 Eg5 KIF 11 siRNA WST 8
27. ol 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 HVJ E D siRNA miRNA 30uM E HVJ E well 37C 5 CO 48 lt Mouse spleen cells PMA ionomycin 1 T cells 7277 Z 97 Cyclophilin B control siRNA 77 Mimic Housekeeping Positive Control 1 PPIB miRNA 96 HVJ E 4 5uL well Cyclophilin B PPIB peptidylprolyl isomerase B 1 Real Time PCR 0120 409 816 FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS S M 002
28. s gt RT PCR mRNA fienomOdbNE i 1 Cell HL 60 Human promyelocytic leukemia cell line ATCC CCL 240 Culture condition 1 x 105cells well 500uL 20 FBS RPMI 1640 Culture plate 24 well plate FALCON 3047 siRNA Very High Potency Hs_CDC2 siRNA QIAGEN Cat No 1027273 Negative control 1 siRNA Ambion Code No AM4639 HVJ E HVJ E 2 5uL D siRNA 10uM E HVJ E well 37C 5 CO 48 HVJFE 4 5uUwel lt 277777 57 HL 60 CDC2 siRNA 859 7277 97 CDC2 B M Real Time PCR 0120 409 816
29. ting miRNA 2 0 08uM E HVJ E well 37C 5 CO 48 HVJ E 4 5uL well Mouse spleen cells PMA ionomycin 1 T cells g 277 Z Mimic Housekeeping Positive Control 1 PPIB 2 50nM Western blotting 2 7 Z 97 Cyclophilin B PPIB peptidylprolyl isomerase B 1 W estern blotting 0120 409 816 FAX 06 6444 7183 ISK E mail HVJ E iskweb co jp 550 0002 1 3 15 URL http www iskweb co jp hvj e No GS M 008 GenomONE Si Technical Data Sheet BALB c mouse primary T cells miRNA transfection Cyclophilin B B actin a b c d e f gd hh GenomONE Si x 1 GenomONE Si Xx 1 2 24p 1 2 GenomONE s
30. tive Quantity normalized 18SrRNA Cyclophilin B miRNA Relative Quantity qRT PCR assays n 3 Cell Viability Pl staining assay Pre stimulated mouse primary T cells Cyclophilin B miRNA Stimulation T cells isolation amp No stimulation SPlenocyte 1 day Transfection 2 days RT PCR mRNA g 777 97 1 Cell Mouse primary T cells isolated from spleen of female C57BL 6 mouse 6 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 1 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX 100 x Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 HVJ E HVJ E 2 5uL D miRNA 2 0 016uM E HVJ E well EE 37 C 5 CO 48 HVJ E 4 5uL well Mouse spleen cells PMA ionomycin 1 T cells
31. tting GenonmONE i 1 Cell Mouse primary T cells isolated from spleen of female BALB c mouse 6 weeks old Stimulation PMA 5nM ionomycin 1ug mL Culture condition 2 x 106 cells well 500uL RPMI 1640 with 10 FBS 1 GlutaMAX 100 x Culture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D D mRNA 200 0 3nM E HVJ E well 37C 5 CO 48 lt Mouse spleen cells PMA ionomycin 1 T cells 2 Z Cyclophilin B control miRNA 400pM RT PCR 75 Western blotting Cyclophilin B PPIB peptidylprolyl isomerase B
32. ulture plate 24 well plate miRNA Mimic Housekeeping Positive Control 1 PPIB Thermo Scientific Cat No CP 002000 01 05 miRIDIAN microRNA Mimic Negative Control 1 Thermo Scientific Cat No CN 001000 01 05 24 well plate HVJ E HVJ E 2 5uL D siRNA miRNA 2uM SiRNA miRNA 10uL E E 5uL HVJ E well 37C 5 CO 48 lt 27Z77 Z 57 Anti CD40 LPS Anti CD40 IL4 mouse primary B cells Mimic Housekeeping Positive Control 1 PPIB miRNA 70 ja Cyclophilin B PPIB peptidylprolyl isomerase B 1 Rea Time PCR HVJ E 4 5uL well ISK 0120 409 816 FAX 06 6444 7183 E mail HVJ E iskweb co jp 550 0002 1

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