Presentation on theme: "Requirement for a new transfection assay for RNAi Each cell has unique transfection efficacy. For successful RNAi, good transfection method is essential."— Presentation transcript:
Requirement for a new transfection assay for RNAi Each cell has unique transfection efficacy. For successful RNAi, good transfection method is essential. One easy way is testing with Fluor-conjugated siRNA. As shown in below, the method is for only transfection but not for RNAi. Another method is testing gene silencing after transfection using qPCR, which demands for cost and labor. A fast and easy transfection assay for RNAi is required. 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.
Transfection control siRNA Materials included Transfection control siRNA (2 nmole) Scramble siRNA (2 nmole) siRNA dissolving buffer The easiest and fastest way for siRNA transfection test Just add to the cell, wait for 48 hours, and count the viable cell The cell toxicity and level of the gene silencing is proportional The target gene of the used siRNA is essential for cell viability For human, mouse, and rat cells
Transfection control siRNA Cell viability of MCF7 in 48 hour Scramble siRNA 1 nM control siRNA 1 nM Gene silencing in 24 hour *All transfection assay was done by a transfection reagent G-Fectin.
The cell toxicity and level of the gene silencing is proportional *All transfection assay was done by a transfection reagent G-Fectin. MCF7
The cytotoxic effect of Transfection control siRNA in mouse and rat cells *All transfection assay was done by a transfection reagent G-Fectin.
G-Fectin 1. A novel transfection reagent developed for RNAi 2. Whole transfection in 10 min on the same day. The fastest and easiest method for transfection. 3. Your gene silencing in 24 hours 4. Lower toxicity 5. Good transfection efficacy 6. Compatible to siRNA. shRNA, and miRNA ml (for 250 X transfection of 24 well plate)
10 min transfection protocol with G-Fectin with G-Fectin 2 ul of G-Fectin siRNA Add the mixture to plate directly Incubate 24 hrs qPCR or Western Add G-Fectin & siRNA to 50 ul of PBS in tube Incubate 10 min at RT 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
G-Fectin has good Transfection efficacy Transfection assay using Vimentin siRNA final 10 nM
Tested cell line list The Transfection efficacy was tested using the Transfection Control siRNA and G-Fectin. Excellent transfection efficacy with 2ul of G-Fectin HeLa, CHO, KB, HT1080, 3T3-L1, COS-1, Caco-2, SKOV3, MDA- MB-468, HEP3B, siHa, MIA-PACA2, PANC1, PLC/PRF/5, MCF7, PC3, C4I, J82, MRC5, HaCat Good transfection efficacy with 4 ul of G-Fectin SNU-638, SNU-368, SNU-475, Noz, OVCA429, T98G, No transfection Jurkat, CEM
G-Fectin has low cytotoxicity All described cells were spitted in 30 % confluency in 36 hour ahead of viable cell counting with treatment of lipo-plexed scramble RNA in final 10 nM.
G-Fectin Kit Materials included Transfection control siRNA (2 nmole) Scramble control siRNA (2 nmole) siRNA dissolving buffer G-Fectin 0.5ml (for 500 X transfection to 24 well) G-Fectin + Transfection Control siRNA The easiest and fastest way for siRNA transfection test Includes all reagents required for transfection and transfection efficacy test Can use to human, mouse, and rat