Takara Xfect RNA Transfection Reagent \1.2 mL

Xfect RNA Transfection Reagent is a complete system for highly efficient transfection of mammalian cells with all types of RNA, including messenger RNA (mRNA), small interfering RNA (siRNA), and single guide RNA (sgRNA).The protocol is simple, and transfections can be carried out entirely in the presence of serum. Xfect RNA Transfection Reagent creates biodegradable nanoparticles when combined with RNA, resulting in very low cytotoxicity.

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells.The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

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 againstluciferaseusing 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.

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.

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.

Functional knockout of the endogenousCD81gene by sgRNA transfection.Panel A.sgRNA targeting the 5’ end of the antisense strand ofCD81was synthesized using the Guide-it sgRNAIn VitroTranscription Kit.Panel B.An HT1080 cell line (2.0 x 105cells) stably expressing Cas9 (HT1080-Cas9) was transfected with 50 pmol of sgRNA targetingCD81, 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 a 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 ofCD81.

Knockout of theluciferasegene by sgRNA transfection.Panel A.Two sgRNAs (sgRNA-A and sgRNA-B) targeting sense and antisense strands ofluciferasewere synthesized using the Guide-it sgRNAIn VitroTranscription Kit.Panel B.3.0 x 105HT1080 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, or 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.