siRNA Delivery to Hard-to-Transfect Cells

By: Lyle Ralston Zhihong Zhang Courtney Corman and Heather Holemon, LSI Issue 24

Lyle Ralston, Zhihong Zhang, Courtney Corman, Heather Holemon,
Research & Development, Biotechnology Division, Sigma-Aldrich

The rapid evolution of RNAi-based gene silencing techniques has provided a number of tools that are being utilized for effective biomarker screening and identification. The use of siRNA to mediate knockdown of targets in these types of applications has been largely limited to the more readily transfectable cell types, which may not be the most relevant cellular model for biomarker detection. Traditional lipid-based siRNA transfection reagents exhibit a limited ability to transfect certain cell types, such as primary, neuronal, differentiated, and non-dividing cells. Sigma’s N-TER™ Nanoparticle siRNA Transfection System is a peptidebased transfection reagent that is specifically designed to bypass these limitations, allowing for the efficient delivery of siRNAs into these historically recalcitrant eukaryotic cell types. Table 1 lists some of the cells that have been successfully transfected using N-TER/siRNA nanoparticles.

3T3-L1
differentiated Mouse, embryonic fibroblast cell line
HEK293T
Human, embryonic kidney cell line
MDA-MB-231
Human, breast adenocarcinoma cell line
A2780
Human, ovarian carcinoma cell line
HeLa
Human, cervical adenocarcinoma cell line
NHA
Human, astrocyte primary cell
A431
Human, ovarian carcinoma cell line
Hepatocyte
Rat, hepatocyte primary cell
NHEK-AD
Human, adult keratinocyte primary cell
A549
Human, lung carcinoma cell line
HepG2
Human, hepatocarcinoma cell line
RAW264.7
Mouse, macrophage cell line
ASPC-1
Human, pancreatic carcinoma cell line
HT-29
Human, colorectal adenocarcinoma cell line
SK-N-SH
Human, neuroblastoma cell line
Astrocytoma
Human, astrocytoma cell line
Huh-7
Human, hepatoma cell line
SW620
Human, colorectal adenocarcinoma cell line
BSMC
Human, bronchial smooth muscle primary cell
HUVEC
Human, umbilical vein epithelial primary cell
THP-1
Human, acute monocytic leukemia cells
C2C12
differentiated Mouse, myoblastoma line
LA-N-2
Human, neuroblastoma cell line
U-87 MG
Human, glioblastomaastrocytoma cell line
C2C12
undifferentiated Mouse, myoblastoma line
MCF-7
Human, breast adenocarcinoma cell line
SK-N-AS
Human, neuroblastoma cell line

The formation of the N-TER Peptide/siRNA complex entails a quick and easy protocol (Figure 1) and the N-TER/siRNA nanoparticles can be assembled in as little as five minutes. The N-TER/siRNA nanoparticles are then diluted into media, in the presence or absence of serum, and added directly to the cells. The entire transfection protocol can be performed in as little as 30 minutes. The flexibility afforded by using a delivery reagent that can perform in media with or without serum permits for optimization of transfection across a wide range of conditions. This versatility allows the N-TER/siRNA nanoparticles to be effectively implemented in the transfection of a wide variety of cell types.

Figure 1.

Figure 1. Figure 1. The workflow for N-TER/siRNA nanoparticle formation and transfection is fast and simple. N-TER/siRNA nanoparticles assemble in as little as five minutes. The nanoparticle is diluted directly into growth medium, in the presence or absence or serum, and added to cells. This whole process can be performed in less than 30 minutes. The cell transfection medium can be left on the cells overnight or removed in as little as 1 hour, greatly accelerating the rate at which siRNA is delivered to the target.


In addition to ease of use, N-TER/siRNA nanoparticles deliver efficient knockdown with minimal cytotoxicity (Figure 2). HeLa and SK-N-SH cells were seeded in 96-well plates and transfected with 10 nM GAPDH siRNA using N-TER and four other transfection reagents. Results demonstrate that transfection with N-TER delivers the most efficient knockdown of GAPDH with the least cellular toxicity in both cell types. N-TER/siRNA nanoparticles can also be used in reverse transfection assays. This method allows the user to eliminate an entire day from the transfection protocol, as cells do not need to be plated on the day prior to transfection. Instead, the nanoparticles are diluted directly into medium containing the trypsinized cells. Results obtained from reverse transfection with N-TER (Figure 3) are similar to those from traditional transfection (Figure 2) and demonstrate highly efficient knockdown with minimal cellular toxicity.

N-TER delivers siRNA into a wide variety of cell types. These include a number of primary cell types and hard-to-transfect cell lines, including differentiated and non-dividing cells.

Figure 2.

Figure 2. Figure 2. N-TER/siRNA nanoparticles efficiently transfects cells with minimal toxicity HeLa (A) and SK-N-SH (B) cells were seeded in 96-well plates at densities of 4,000 and 5,000 cells per well, respectively. Cells were then incubated at 37 °C, 5% CO2 for approximately 20 hours. The cells were subsequently transfected with 10 nM GAPDH siRNA, and complexed with N-TER and four other transfection reagents (C1 – C4). The cells were then incubated at 37 °C, 5% CO2 for 24 hours. Following transfection, the cells were harvested and assayed for gene expression and viability. Gene expression assays were performed using the QuantiGene® branched DNA (bDNA) assay. Viability assays were performed using Promega’s CellTiter-Glo™ Luminescent Cell Viability Assay.


Figure 3.

Figure 3. N-TER/siRNA nanoparticles can be used in reverse transfection assays. Three methods were tested for efficacy of delivery of 20 nM GAPDH siRNA into HeLa cells (A). In method 1, 10 μL of the N-TER/siRNA nanoparticle was spotted in the bottom of each well, then 100 μL of trypsinized cells was added. In method 2, the nanoparticle was diluted into 50 μL of growth medium and added to each well, then 50 μL of trypsinized cells was added. In method three the nanoparticle was diluted directly into the medium containing the trypsinized cells and then 100 μL of the nanoparticle-cell mixture added to each well. Method 3 was used to transfect HT-29 cells (B) at three different siRNA concentrations and three different cell densities. In both cases, cells were incubated at 37°C, 5% CO2 for 24 hours then harvested and assayed for gene expression and viability.


N-TER/siRNA nanoparticles rapidly deliver the siRNA into cells (Figure 4). Significant knockdown can be seen with as little as 30 minutes of exposure to the nanoparticles. In Figure 4, HeLa cells were transfected with 10 nM GAPDH siRNA using the standard N-TER protocol. After the indicated times, the medium containing N-TER/siRNA nanoparticles was replaced with fresh medium. Cells were harvested 24 hours later. Results show that significant knockdown is seen after a 30-minute exposure to the N-TER/siRNA nanoparticles. Knockdown increases as exposure time increases. Rapid delivery allows the user to optimize transfection conditions, finding a balance that maximizes target knockdown while minimizing cellular toxicity.

Figure 4.

Figure 4. N-TER/siRNA nanoparticles quickly deliver siRNA into cells. HeLa cells were transfected with 10 nM GAPDH siRNA using the standard transfection procedure described in Figure 2. Cells were incubated with transfection medium for the indicated times. At the end of the indicated incubation periods, the transfection medium was replaced with fresh growth medium. After 24 hours, cell were harvested and assayed for gene expression.


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