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Ⅵ. G-Fectin (transfection reagent) RNAi transfection 전용 reagent. Transfection 전 과정 10분 이내 완료. 24시간 후 gene silencing 확인 가능. 우수한 transfection 효율 낮은 toxicity. sirna와 shrna, mirna에 모두 적용 가능. 가격 : 150,000 원/0.5 ml (for 250-500 transfection of 24 well plate) Fig 1. G-Fectin has good transfection efficacy *Transfection assay using Vimentin sirna final 10 nm Ⅵ. G-Fectin (transfection reagent) 2
Fig 2. G-Fectin has low cytotoxicity * All described cells were spitted in 30% confluency in 48h ahead of viable cell counting with treatment of lipo-plexed scramble RNA in final 10 nm. <Transfection control sirna validated cell types> Excellent transfection efficacy Good transfection efficacy Cell line HeLa, CHO, KB, HT1080, 3T3-L1, COS-1, Caco-2, SKOV3, MDA-MB-468, Hep3B, HepG2, Her-7, siha, MIA-PACA2, PANC1, PLC/PRF/5, MCF7, PC3, C4I, J82, MRC5, HaCat, HCT119, mppcp (porcine), PLOG/NW (porcine), rath9c2, SNU-484, TE10, AGS SNU-638, SNU-368, SNU-475, Noz, OVCA429, T98G G-Fectin 1 ul 2 ul No transfection Jurkat, CEM N/A Ⅵ. G-Fectin (transfection reagent) 3
G-fectin 을 이용한 10 min. Transfection Protocol G-Fectin으로 동물 세포에 sirna, mirna, shrna를 transfection 하는 방법으로 24-well plate를 기준으로 합니다. STEP 1. Preparation of Transfection Mixture 1. 각 E. tube에 shrna/sirna를 원하는 용량을 분주한다. (예; 만약 final 10 nm 농도로 transfection 하기 위해서는 5uM 농도의 RNA stock 을 final media volume 500ul 을 기준으로 1 ul 넣어준다.) 2. 위의 tube 에 PBS 50ul 과 G-fectin 1 또는 2ul 를 첨가해서 잘 섞은 후 10 분 동안 incubation 한다. STEP 2. Cell plating and Application to Cells 1. 10min 동안 24well plate에 cell을 20~30% confluency로 plating한다. 2. 10min후 transfection mixture를 준비된 cell에 바로 넣어준다. 타 제품과 달리 이전날 세포를 split하거나 transfection mixture 를 일정시간 경과 후 제거할 필요가 전혀 없음. 3. 24-48 시간 이후 RNAi validation G-Fectin 도착 즉시 4-8 보관. <Cell culture volume에 따른 transfection condition> Culture Plate Vol. of total medium RNAi Mixture (RNA; 10-30 nm) Vol. of cell suspend (Surface area per well) Vol. of 1xPBS G-Fectin 96 well 100 ul (0.3 cm 2 ) 90 ul 10 ul 0.5-1 ul 24 well 500 ul (2 cm 2 ) 450 ul 50 ul 1-2 ul 12 well 1 ml (4 cm 2 ) 900 ul 100 ul 2-4 ul 6 well 2 ml (10 cm 2 ) 1.8 ml 200 ul 4-6 ul 60 mm 5 ml (20 cm 2 ) 4.5 ml 500 ul 10-15 ul 100 mm 15 ml (60 cm 2 ) 13.5 ml 1.5 ml 30-50 ul Ⅵ. G-Fectin (transfection reagent) 4
Ⅶ. Easy RNAi package RNAi를 위한 새로운 transfection 방법 필요. - 각각의 세포는 서로 다른 transfection 효율을 가짐. - Fluor-conjugated sirna를 이용한 실험은 transfection 유무만 확인 가능함 (Fig. 1). - transfection 후 gene silencing을 확인하는 qpcr 방법은 많은 시간과 비용 이 소요됨 (Fig. 2). Fig. 1 Before(left) and after(right) transfection of CEM cell with FITC-conjugated Vimentin sirna Fig. 2 Total RNA sample was prepared after the transfection and used for qpcr for test the silencing of target gene. Unlike HeLa, there was no sign of gene silencing even thought indication of good transfection by the FITC-conjugated sirna. Ⅵ. G-Fectin (transfection reagent) 5
A. Easy sirna transfection assay kit sirna transfection test를 위한 가장 쉽고 빠른 방법. 세포에 transfection 후 48시간 이내 세포생존 육안 관찰. 세포독성과 유전자 silencing의 효과는 비례. 사용된 sirna의 표적유전자는 세포생존에 필수적임. 모든 human, mouse, 그리고 rat cells에 공통적으로 사용가능. 195,000원/tube (for more than 100 test) Materials included - Transfection control sirna (for more than 100 test) - Scramble control sirna (for more than 100 test) - sirna dissolving buffer Protocol STEP 1. Preparation of Transfection Mixture 1. Tube에 500 ul의 물을 첨가하여 녹인다. (1 um) 2. Control sirna 2.5 ul를 분주한다. (final 농도 5nM) 3. 위의 tube에 PBS 50ul과 G-Fectin 1 또는 2ul 를 첨가해서 잘 섞은 후 10 분 동안incubation한다. STEP 2. Cell plating and Application to Cells 1. 10min 동안 24well plate에 cell을 20~30% confluency로 plating한다. 2. 10min후 transfection mixture를 준비된 cell에 바로 넣어준다. 타 제품과 달리 전날 세포를 split하거나 transfection mixture 를 일정시간 경과 후 제거할 필요가 전혀 없음. 3. 24-48 시간 이후 RNAi validation Ⅵ. G-Fectin (transfection reagent) 6
Transfection test sirna Cell viability of MCF7 in 48 hour Scramble sirna 1nM Transfection test sirna 1nM Gene silencing in 24 hour Fig 1. All transfection assay was done by a transfection reagent, G-Fectin. Ⅵ. G-Fectin (transfection reagent) 7
Fig 2. The cell toxicity and level of the gene silencing is proportional (MCF7) * All transfection assay was done by a transfection reagent G-Fectin Fig 3. The cytotoxic effect of transfection control sirna in mouse and rat cells * All transfection assay was done by a transfection reagent G-Fectin Ⅵ. G-Fectin (transfection reagent) 8
B. G-Fectin kit 가장 쉽고 빠른 sirna Transfection test kit. Transfection reagent G-Fectin 과 transfection efficacy test를 위한 Transfection test sirna 포함. 모든 human, mouse 그리고 rat cells에 공통적으로 사용가능. 가격 : 290,000 원 / kit (부가세별도) Materials included - Transfection test sirna (for more than 100 test) - Scramble sirna (for more than 100 test) - sirna dissolving buffer - G-Fectin 0.5 ml (for 250-500xtrasnfection to 24 well) Ⅵ. G-Fectin (transfection reagent) 9
G-Fectin Kit Transfection Protocol G-Fectin으로 동물 세포에 sirna, mirna, shrna를 transfection 하는 방법으로 24-well plate를 기준으로 합니다. Protocol STEP 1. Preparation of Transfection Mixture 1. Tube에 500 ul의 물을 첨가하여 녹인다. (1 um) 2. Test sirna 2.5 ul를 분주한다. (final 농도 5nM) 3. 위의 tube에 PBS 50ul과 G-Fectin 1 또는 2ul 를 첨가해서 잘 섞은 후 10 분 동안 incubation 한다. STEP 2. Cell plating and Application to Cells 1. 10min 동안 24well plate에 cell을 20~30% confluency로 plating한다. 2. 10min후 transfection mixture를 준비된 cell에 바로 넣어준다. 타 제품과 달리 이전날 세포를 split하거나 transfection mixture 를 일정시간 경과 후 제거할 필요가 전혀 없음. 3. 24-48 시간 이후 RNAi validation G-Fectin 도착 즉시 4-8 보관. <Cell culture volume에 따른 transfection condition> Culture Plate Vol. of total medium RNAi Mixture (RNA; 10-30 nm) Vol. of cell suspend (Surface area per well) Vol. of 1xPBS G-Fectin 96 well 100 ul (0.3 cm 2 ) 90 ul 10 ul 0.5-1 ul 24 well 500 ul (2 cm 2 ) 450 ul 50 ul 1-2 ul 12 well 1 ml (4 cm 2 ) 900 ul 100 ul 2-4 ul 6 well 2 ml (10 cm 2 ) 1.8 ml 200 ul 4-6 ul 60 mm 5 ml (20 cm 2 ) 4.5 ml 500 ul 10-15 ul 100 mm 15 ml (60 cm 2 ) 13.5 ml 1.5 ml 30-50 ul Ⅵ. G-Fectin (transfection reagent) 10
Ⅷ. Easy RNAi Protocol The 10min transfection protocol by G-Fectin. Whole transfection in 10 min on the same day. The fastest and easiest method for transfection. Your gene silencing in 24 hours. 1-2 ul of G-Fectin Genolution sirna Add the G-fectin & sirna to 50ul of PBS in tube 10 min transfection protocol with G-Fectin 50 ul of PBS Incubate 10 min at RT Add the mixture to plate directly Cells seed on plate Detached the cell and add 450 ul of media (5 x 10 4 cells/ well) to 24 well plate No cell splitting is required in previous day Incubate 24 hrs qpcr Fig. 1 Transfection protocol Ⅵ. G-Fectin (transfection reagent) 11
Fig 2. RNA prep protocol Ⅵ. G-Fectin (transfection reagent) 12
Easy Protocol for Quantitative Real time PCR <Real time RT PCR condition> 2x One step SYBR RT-PCR buffer - I Takara Ex taq HS (5U/ul) - II Primescript RT enzyme mix - III PCR F- primer (10uM) PCR R- primer (10uM) Total RNA (10ng/ul) RNase free water 12.5 ul 0.5 ul 0.5 ul 0.5 ul 0.5 ul 5 ul 5.5 ul Total 25 ul Machine : SMARTcycler (Cepheid) Stage 1: Reverse transcription (42 5min, 95 10sec) Stage 2: PCR reaction (95 5sec, 60 20sec) - 50cycles Stage 3: Dissociation (melting) from 60 to 95 Intercalate <EXAMPLE> Melting Curve Fig 3. Real Time PCR protocol Ⅵ. G-Fectin (transfection reagent) 13
Cell culture dish information 1. Flask information 2. Dish information Ⅵ. G-Fectin (transfection reagent) 14
3. Well information Ⅵ. G-Fectin (transfection reagent) 15
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