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Transfect all RNA types with Xfect RNA Transfection Reagent

The Xfect RNA Transfection Reagent is compatible with all types of RNA, including mRNA, single guide RNA (sgRNA), microRNA, and siRNA. The protocol is simple, and transfections can be carried out entirely in the presence of serum. Unlike most transfection reagents, the Xfect RNA Transfection Reagent has very low cytotoxicity. It combines with RNA to create biodegradable nanoparticles, which allow efficient mammalian cell transfection without any of the harmful side effects.

Transfection of messenger RNA with this reagent allows you to achieve rapid gene expression and avoid the risk of genomic integration that accompanies DNA transfection. Xfect RNA Transfection Reagent also efficiently delivers single guide RNAs (sgRNA) for CRISPR/Cas9-mediated gene editing.

The Xfect RNA Transfection Reagent can be used together with the Xfect Transfection Reagent to cotransfect mammalian cells with both DNA and RNA.

siRNA Transfection  

Why use the Xfect RNA Transfection Reagent to transfect siRNA oligos?

  • It's effective—get better knockdown than with the leading competitor
  • It's easy—no optimization necessary
  • It's nontoxic—Xfect won't kill your cells

Effective gene silencing using transfected siRNA oligos

Having good siRNA oligos is not enough if you can't deliver those siRNAs effectively since your experiment will fail. The Xfect RNA Transfection Reagent was developed to maintain a very low cytotoxicity profile while delivering high rates of gene silencing. The ratio of oligo to reagent is pre-optimized, so you don't have to waste time on preliminary experiments. Transfecting siRNA with Xfect RNA Transfection Reagent has been shown to result in successful knockdown, e.g., a 95% decrease in luciferase activity (Figure 1).

Successful knockdown in primary cells and cell lines treated with siRNA

Figure 1. Successful knockdown in primary cells and cell lines treated with siRNA. HeLa cells (2.0 x 105), Human Dermal Fibroblasts (NHDF) cells (6.0 x 104), and Mesenchymal Stem Cells (MSCs) (5.0 x 104) were plated in 12-well plates and transfected with 50 pmol of siRNA against luciferase using Xfect RNA Transfection Reagent. All three cell types were also transfected with 1 µg of pCMV-Luc using the Xfect Transfection Reagent. Luciferase assays were performed 48 hours post-transfection. For control samples, cells were transfected with pCMV-Luc but without the siRNA. We observed a dramatic (>95%) decrease in luciferase activity in all the cells treated with siRNA.

mRNA Transfection  

Rapid gene expression using transfected mRNA

Transfection with messenger RNA using the Xfect RNA Transfection Reagent allows for rapid gene expression (within 20–24 hr after transfection), without the risk of genomic integration that comes from transfection with DNA. This is demonstrated quantitatively (Figure 2) and visually (Figure 3) for GFP mRNA, and quantitatively (Figure 4) for firefly luciferase mRNA.

Expression of GFP from primary cells and cell lines transfected with mRNA

Figure 2. Expression of green fluorescent protein (GFP) from transfection with mRNA into primary cells and cell lines. HeLa cells (2.0 x 105), HEK 293 cells (1.5 x 105), Human Dermal Fibroblasts (NHDF) cells (6.0 x 104), Mesenchymal Stem Cells (MSCs) (5.0 x 104), Jurkat Cells (3.0 x 105), and KBM7 cells (3.0 x 105) were plated in 12-well plates and transfected with 1 µg of mRNA encoding GFP with 5 µl of Xfect RNA Transfection Reagent. 20 hours later, cells were analyzed by flow cytometry and the % GFP-positive cells and the Mean Fluorescence Intensity (MFI) were determined.

 

Figure 3. GFP is expressed in HeLa cells, NHDF cells, and MSCs. HeLa cells (2.0 x 105), Human Dermal Fibroblasts (NHDF) cells (6.0 x 104), and Mesenchymal Stem Cells (MSCs) (5.0 x 104) were plated in 12-well plates and treated with 1 µg of mRNA encoding GFP with 5 µl of Xfect RNA Transfection Reagent. 20 hours later, cells were imaged using an epifluorescent microscope. Images of HeLa cells were taken at 20X magnification. Pictures of NHDF cells and MSCs were taken at 40X.

 

Xfect MicroRNA Transfection Reagent successfully transfects firefly luciferase (Fluc) messenger RNA into HeLa cells.

Figure 4. Xfect RNA Transfection Reagent successfully transfects firefly luciferase (Fluc) messenger RNA into HeLa cells. Fluc messenger RNA (0.5 μg and 1.0 μg) was transfected into HeLa cells using Xfect RNA Transfection Polymer according to the protocol described above. 24 hours after transfection, luciferase activity (Relative Light Units; RLU) was measured using a luciferase assay.

miRNA Transfection  

Efficient microRNA transfection

Xfect RNA Transfection Reagent provides high levels of transfection efficiency and cell viability for six different popular cell lines (Figure 5).

 

Efficient transfection of microRNA into 6 mammalian cell lines.

Figure 5. Efficient transfection of microRNA using the Xfect RNA Transfection Reagent. A FAM-labeled microRNA mimic was transfected into six popular cell lines using the Xfect RNA Transfection Reagent according to the protocol. 24 hr post-transfection, cells were harvested, and transfection efficiency was determined by flow cytometry. Cell viability was also measured using Trypan Blue staining. Data represents the average of three independent transfections for each cell line.

sgRNA Transfection  

Efficient Delivery of sgRNAs

Xfect RNA Transfection Reagent can efficiently deliver single guide RNAs (sgRNAs) for CRISPR/Cas9-mediated gene editing. sgRNA transfection using this reagent resulted in a functional knockout of the endogenous CD81 gene (Figure 6) and a robust (>95%) knockout of the luciferase gene (Figure 7).

Functional knockout of the endogenous CD81 gene by sgRNA transfection

Figure 6. Functional knockout of the endogenous CD81 gene by sgRNA transfection. Panel A. sgRNA targeting the 5' end of the antisense strand of CD81 was synthesized using the Guide-it sgRNA In Vitro Transcription Kit. Panel B. An HT1080 cell line (2.0 x 105 cells) stably expressing Cas9 (HT1080-Cas9) was transfected with 50 pmol of sgRNA targeting CD81, either once or twice (lower graph), using the Xfect RNA Transfection Reagent. Seven days later, cells were immunostained with a CD81 antibody (Ab) conjugated to an FITC fluorophore and analyzed by flow cytometry. The percentage of cells that did not bind CD81 was calculated. A control sample, comprised of HT1080-Cas9 cells, was analyzed by flow cytometry, either without (top, left graph) or with (top, right graph) the CD81 antibody. Both single and double transfection with sgRNA resulted in a substantial increase in cells that did not bind CD81, indicating successful CRISPR/Cas9-mediated knockout of CD81.

  

Knockout of the luciferase gene by sgRNA transfection

Figure 7. Knockout of the luciferase gene by sgRNA transfection. Panel A. Two sgRNAs (sgRNA-A and sgRNA-B) targeting sense and antisense strands of luciferase were synthesized using the Guide-it sgRNA In Vitro Transcription Kit. Panel B. 3.0 x 105 HT1080 cells stably expressing Cas9 (HT1080-Cas9) were plated in 12-well plates and transfected with 1 µg of pCMV-Luc (using the Xfect Transfection Reagent) and 200 ng of either sgRNA-A, sgRNA-B, or 100 ng each of both sgRNAs (using the Xfect RNA Transfection Reagent). 48 hours post-transfection, luciferase assays were performed. Robust knockout (>95%) of luciferase activity was observed with each sgRNA individually and in combination with each other.

Summary  

Xfect RNA Transfection Reagent has been shown to be effective for transfecting all types of RNA, including siRNA, mRNA, microRNA, and sgRNA. Using this reagent with primary cells and cell lines resulted in successful knockdown when transfecting siRNA oligos, and allowed for rapid gene expression when transfecting mRNA. Xfect RNA Transfection Reagent also provided efficient microRNA transfection and enabled effective gene knockouts when it was used to deliver sgRNAs for CRISPR/Cas9-mediated gene editing. The high transfection efficiency, low cytotoxicity, and versatility of this reagent make it a powerful tool for RNA transfection.

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