Applications: Stem Cells

RNA Transfection for Stem Cell Applications

Transfect mRNA using TransIT®-mRNA Transfection Kit for Stem Cell Reprogramming

  • Low Cellular Toxicity - Perform repeated transfections easily while maintaining cell density
  • High Efficiency Delivery - Achieve RNA delivery in hard to transfect cells including stem cells
  • Serum Compatible - Perform transfections in the presence of serum which eliminates the need for a media change and maintains cellular health
  • Deliver Various Sizes of RNA - Ideal for specialized applications, such as viral production and protein expression from mRNA
TransIT®-mRNA (1:1:1)
RNAiMAX (4:1)
Stemfect™ (2:1)
TransIT-mRNA day 1 TransIT-mRNA day 5 TransIT-mRNA day 9 TransIT-mRNA day 14 TransIT-mRNA day 14 cells
RNAiMax day 1 RNAiMax day 5 RNAiMax day 9 RNAiMax day 14 RNAiMax day 14 cells
Stemfect day 1 Stemfect day 5 Stemfect day 9 Stemfect day 14 Stemfect day 14 cells
Click on the red days to view images

High Efficiency and Low Toxicity Transfection Following 14 Consecutive Transfections with TransIT®-mRNA Transfection Kit. Repeated daily transfections were performed in the same population of BJ fibroblasts using TransIT®-mRNA Transfection Kit, Lipofectamine® RNAiMAX (Life Technologies) and Stemfect™ RNA Transfection Kit (Stemgent) -  with a capped and polyadenylated EGFP mRNA incorporating pseudouridine and 5mC modified bases (Trilink Biotechnologies, Inc.).  Multiple reagent-to-RNA ratios were tested and the optimal ratio is represented. Transfections were performed in 12-well plates using the indicated reagent-to-RNA ratios to deliver 1 µg of RNA. GFP and phase contrast images were taken in the same field of view everyday after transfection. Representative images are shown at Days 1, 5, 9 and 14 post-transfection. Cells transfected with TransIT®-mRNA Transfection Kit exhibit the highest transfection efficiency with minimal cytotoxicity. Lipofectamine® RNAiMAX has low transfection efficiency, whereas Stemfect™ RNA Transfection kit displays significant cytotoxicity. Transfection efficiency and cell count measurements using flow cytometry are shown below.

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High Efficiency and Low Toxicity Transfection Following 14 Consecutive Transfections with TransIT-mRNA Transfection Kit.

TransIT®-mRNA Transfection Kit yields Higher Efficiency and Cell Viability following 14 Consecutive Transfections Compared to Lipofectamine® RNAiMAX and Stemfect™ RNA Transfection Kit. Experimental details on repeated transfections are described above. Transfection efficiency was measured by flow cytometry on a Guava® easyCyte™ 5HT following 14 consecutive daily transfections (blue bars). Cell viability was determined using cell counts measured during flow cytometry (black line grey bars). Error bars represent the standard deviation of triplicate wells.

Our lab recently used theTransIT-mRNA Transfection Kit to show that intracellular delivery of HPLC-purified and pseudouridine-containing mRNA can translate very efficiently without immune activation which is ideal for mRNA-based gene therapy applications. TransIT®-mRNA further facilitated this work through low toxicity transfections of HEK 293T, human dendritic cells (DCs) and primary keratinocytes. (Karik√≥ et al. Nucleic Acids Research, 39:e142, 2011)
Dr. Katalin Kariko
University of Pennsylvania - Department of Neurosurgery

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