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TransIT-X2® Dynamic Delivery System for Broad Spectrum Transfection

An advanced system for delivery of plasmid DNA, siRNA/miRNA and CRISPR/Cas9 components

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Experience the transfection breakthrough. Achieve superior transfections with an innovative polymeric system that efficiently delivers both DNA and RNA out of the endosome and into the cytoplasm overcoming a critical barrier to nucleic acid delivery. The TransIT-X2® Dynamic Delivery System gives researchers:

icon Efficiency – Exceptional Broad Spectrum Transfection
icon Versatility – Cutting Edge Delivery of Plasmid DNA and small RNAs (siRNA, miRNA and CRISPR guide RNA)
icon Technology – Novel, Non-Liposomal, Polymeric Delivery

Learn more about using TransIT-X2® for CRISPR/Cas9 genome editing

 

TransIT-X2® System Enables Superior Gene Expression in a Variety of Cell Types

‡ Cell types with >2-fold greater expression in head-to-head comparisons
Click here for cell-type specific transfection protocol recommendations
 

TransIT-X2® Dynamic Delivery System Enables Superior Transfection in a Variety of Cell Types. TransIT-X2® Dynamic Delivery System and Lipofectamine® 2000 Transfection Reagent were used to transfect plasmid DNA encoding luciferase into 41 different cell types at three reagent-to-DNA ratios. Luciferase expression was compared at 24 hours post-transfection using a standard luciferase assay. Head-to-head comparisons at optimized ratios illustrate superior or equal luciferase expression using TransIT-X2® in 36 of 41 cell types; 17 cell types that had expression levels 2-fold higher are denoted with ‡.

Figures and Data

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TransIT-X2 Dynamic Delivery System Outperforms Lipofectamine Reagents

TransIT-X2® Dynamic Delivery System Outperforms Lipofectamine® Reagents. A549 (A) or MDCK (B) cells were transfected with luciferase encoding plasmid DNA using either TransIT-X2® (Mirus Bio), Lipofectamine® 2000 (Thermo Fisher Scientific) or Lipofectamine® 3000 (Thermo Fisher Scientific) for 24 hours at indicated reagent-to-DNA ratios or reagent-to-P3000™-to-DNA ratio. Transfection was measured by luciferase activity using a conventional assay. Cytotoxicity was assessed by quantifying the LDH released from the cytosol of damaged cells compared to cells alone. Higher transgene expression and lower cytotoxicity was observed in cells transfected with TransIT-X2® at optimal ratios compared to cells transfected with Lipofectamine® 2000 or Lipofectamine® 3000.

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Visualization of High GFP Expression Using TransIT-X2 Dynamic Delivery System

Visualization of High GFP Expression Using TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, HepG2, LNCaP, MDCK, PC12, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF). Transfections were performed in 35 mm MatTek dishes using 4-8 µl of TransIT-X2® to deliver 2 µg of DNA. Images (32X) were captured at 48 hours post-transfection using a Zeiss Axiovert S100 inverted fluorescence microscope.

* indicates primary cell types

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High GFP Transfection Efficiency in Multiple Cell Lines and Primary Cells Using TransIT-X2 System

High GFP Transfection Efficiency in Multiple Cell Lines and Primary Cells Using TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, Hep G2, MDCK, LNCaP, PC-12, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF). Transfections were performed in 96-well plates using 0.2-0.4 µl of TransIT-X2® to deliver 0.1 µg of DNA (2:1, 3:1 or 4:1 reagent: DNA ratio). Triplicate wells were assayed 48 hours post-transfection using guava easyCyte™ 5HT Flow Cytometer.

*indicates primary cell types

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Functional Co-transfection of Plasmid DNA and siRNA Using TransIT-X2 System

Functional Co-transfection of Plasmid DNA and siRNA Using the TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid Cy®5 labeled DNA encoding nuclear YFP and Cy®3 labeled siRNA into HeLa cells. Transfection was performed in 6-well plates with Poly-L-Lysine (PLL) coated coverslips using 4 µl of TransIT-X2® to deliver 2 µg of DNA and 25 nM siRNA (2:1 reagent:DNA ratio). Actin cytoskeleton was stained using Alexa Fluor® 350 Phalloidin. Images (63X) were captured at 24 hours post-transfection using a Nikon A1R confocal microscope. Image key: yellow (nuclear YFP), blue (Cy®5 labeled DNA), red (Cy®3 labeled siRNA), green (actin cytoskeleton).

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TransIT-X2 Dynamic Delivery System Achieves Higher Knockdown than Lipofectamine 2000

TransIT-X2® Dynamic Delivery System Achieves Higher Knockdown than Lipofectamine® 2000. TransIT-X2® Dynamic Delivery System and Lipofectamine® 2000 Transfection Reagent were used to transfect siRNA targeting endogenous proteins – GAPDH and AHA1 or to deliver a non-targeting siRNA control in normal human dermal fibroblasts (NHDF). Cells were transfected in a 6-well plate using 4 µl of TransIT-X2® or 6 µl of Lipofectamine® 2000 and 25 nM siRNA according to each manufacturer's protocol. The amount of GAPDH or AHA1 mRNA was measured relative to 18s rRNA levels using qRT-PCR and then normalized to the mRNA levels of the non-targeting control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells.

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Effective miRNA Delivery Using TransIT-X2 Yields Decreased Levels of PTK9 mRNA

Effective miRNA Delivery Using TransIT-X2® Dynamic Delivery System Yields Decreased Levels of PTK9 mRNA. TransIT-X2® Dynamic Delivery System and Lipofectamine® 2000 Transfection Reagent were used to transfect Pre-miR™ hsa-miR-1 miRNA Precursor or mirVana™ miRNA mimic, miR-1, both known to decrease PTK9 mRNA levels. A Pre-miR™ negative control was also transfected to assess baseline mRNA levels. T47D cells were transfected in a 12-well plate using 3 µl of TransIT-X2® or Lipofectamine® 2000 and 50 nM miRNA according to each manufacturer's protocol. The amount of PTK9 mRNA was measured relative to 18s rRNA levels using qRT-PCR and then normalized to the mRNA levels of the negative control, 48 hours post-transfection.  Error bars represent the standard deviation of triplicate wells.

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High Efficiency CRISPR Genome Editing with TransIT-X2

Efficient Genome Editing with Cas9 Plasmid DNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding pDNA (MilliporeSigma) and 50nM PPIB targeting 2-part gRNA (Dharmacon) using TransIT-X2® Dynamic Delivery System (2 µl/well of a 24-well plate, Mirus Bio).  A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

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TransIT-X2 Outperforms Lipofectamine for RNP Delivery.

TransIT-X2® Outperforms Lipofectamine® for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB (cyclophilin B) targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 and U2OS cells using TransIT-X2® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine® Cas9 Plus™ Reagent, ThermoFisher) or Lipofectamine® RNAiMAX (1.5 µl/well, ThermoFisher) or Lipofectamine® 3000 (1.5 µl/well and 1 µl/well of P3000™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM or 12 nM) were tested with 6 nM Cas9 protein (PNA Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

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Storage Conditions:
All Configurations: Store at -20°C

Product Guarantee:
All Configurations: 1 year

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Testimonials

I was recently tasked with developing a CRISPR protocol for primary and bone-derived cell lines. TransIT-X2® was simple to use, 2-3 times better for transfection and much gentler on my cells than other products! I feel I have hit the jackpot and have already passed this exciting information on to my colleagues.

The TransIT-X2® Dynamic Delivery System outperformed all other transfection reagents we have tested for DNA transfection of our C2C12 mouse myoblast cell line. In addition, TransIT-X2® was also less toxic.

We work on non-small cell lung cancer (NSCLC) which is an adherent cell culture line. Previously, we have tested many transfection products from several companies without much success, but the TransIT-X2® Dynamic Delivery System works very well with NSCLC using my protocol.

We recently tested the TransIT-X2® Dynamic Delivery System head-to-head against Lipofectamine® 2000 for DNA transfection of NIH-3T3 fibroblasts and the breast cancer cell line ZR-75-1. We observed higher efficiency and less toxicity when using TransIT-X2®. We are also pleased to hear that TransIT-X2® will be offered in similar volume configurations to Lipofectamine® 2000.

We were pleased with the performance and low toxicity of the TransIT-X2® Dynamic Delivery System when transfecting our renal carcinoma cell line 786-0.

A549 TransIT-X2

The TransIT-X2® Dynamic Delivery System performed better than our regular transfection reagent (Polyjet™) for delivering DNA into the hard to transfect A549 cell line. TransIT-X2® was able to show protein expression compared to Polyjet™ which failed to produce detectable levels of protein containing V5 tag.