Self-delivering siRNA for difficult-to-transfect cells transfection-free gene silencing with Accell™ 2.0 siRNA

Peer-reviewed publications demonstrate effective silencing in numerous cell types
Human Suspension Cell Lines	BDCM, IM-9, Jurkat, K662, THP-1, Jeko-1 and Mino MCL
Human Adherent Cell Lines	A-375, ARPE-19, CaCo-2, CG4, DLD-1, DU-145, EC52, GTM-3, MR32, LAN5, LNCaP, MIA PaCa-2, NCI/ADR-RES, OVCAR-3, OVCA-420, PGA1, PNT2, SH-SY5Y, SK-N-BE and SK-N-SH neuroblastoma, SK-BR3, SKOV3, TE15, U251, U87MG, U937
Mouse Adherent Cell Lines	ES-D3, H9c2, LbT2, LNK, mouse oviduct epithelial cells, mProx24, PC-12
Human Differentiated Stem Cells	Adipocytes and osteoblasts from hMSC
Human Primary Cells	HUVEC, HUASMC, hMSC, PBMC, astrocytes, hypothalamic neurons, T cells, NK cells, macrophages, immortalized B cells, esthesioneuroblastoma cells, hepatocytes, bronchial smooth muscle cells
Mouse and Rat Primary Cells	Cortical neurons, hippocampal neurons, striatal neurons, Naïve CD4+ T cells, LN T cells, D1 T cells, RAW264.7 macrophages, hepatocytes, neonatal ventricular cardiomyocytes, fibroblasts derived from genetically-modified mice, bone marrow-derived DC, NOD CD4+ CD25- splenic cells, NOD BMMs, VSMCs, somatolactotroph GH3, McArdle 7777 cells (hepatoma cell line), osteoclast precursors, calvarial (cranial) organ, pituitary cell types derived from primary tumors
In vivo Animal Applications	Please contact Scientific Support for guidance

 

---

 

Application of Accell 2.0 siRNA simplifies targeted gene knockdown

1. Combine Accell 2.0 siRNA with Accell siRNA delivery media (or other reduced serum media).
2. Add Accell 2.0 delivery mix directly to cells and incubate for 72 hours.
   Figure created in with BioRender.com

 

---

 

Accell 2.0 siRNA maintains performance of gene target knockdown set by Accell siRNA in neurons derived from induced WTC-11 iPSC cells

 

Characterization of target mRNA knockdown by Accell and Accell 2.0 siRNAs in cultured neurons. Data are shown relative to neurons treated with non-targeting control (NTC) siRNA. Experimental schematic (A) and relative gene expression in WTC-11-derived neurons (B).  Neurons derived from WTC-11 iPSCs were cultured in differentiation medium for 14 days, with daily medium replenishment. On day 14, 50 µL of medium was removed and replaced with 50 µL fresh medium containing 2 µM Accell or Accell 2.0 pooled siRNA designs (4 siRNA designs at 0.5 µM each) or 2 µM Accell PPIB control (Cat# D-001920-01), resulting in a final siRNA concentration of 1 µM in all conditions. An identical concentration of Accell Non-targeting Control siRNA #1 (D-001910-01) was used for each target NTC condition. siRNA mixtures were replenished by half-medium change on differentiation days 16 and 18, for a total of three siRNA additions. Total RNA was extracted on differentiation day 20 for downstream processing. Figure A is created with BioRender.com.

 

---

 

Performance of target mRNA knockdown in matrix-free 3D spheroids is maintained with Accell 2.0 siRNA

Characterization of Accell and Accell 2.0 siRNA-mediated mRNA knockdown in ECM-free HCT-116 spheroids. Control, PTEN (A), and PPP2R1B (B) mRNA expression were quantified in HCT-116 spheroids after 144 hours of siRNA treatment. (C) Dosing timeline for Accell 2.0 siRNA delivery. Spheroids were generated by seeding 3,000 HCT-116 cells into ECM-free, 96-well ultralow attachment round-bottom plates in HCT-116 medium (McCoy’s 5A medium supplemented with 10% FBS and 2 mM L-glutamine). After two days, 50% of the medium was removed and replaced with 2 µM Accell or Accell 2.0 single or pooled siRNA designs (4 siRNAs at 0.5 µM each) for a final concentration of 1 µM siRNA. Matching concentrations of Accell Non-targeting Control siRNA #1 (D-001910-01) or Accell PPIB control siRNA (D-001920-01) were added on the same schedule. After three rounds of siRNA replenishment, spheroids were lysed, total nucleic acid was collected, and RT-qPCR was used to measure target mRNA abundance. Data are shown relative to target expression in spheroids treated with non-targeting control siRNA. Figure C is created with BioRender.com.

 

---

 

Target protein knockdown in matrix-free 3D spheroids using Accell 2.0 siRNA

Characterization of Accell siRNA-mediated cyclophilin B (PPIB) target protein knockdown in ECM-free HCT-116 spheroids. Data are presented relative to expression in spheroids treated with non-targeting control (NTC) siRNA. (A) Western blot and (B) quantification of Cyclophilin B protein expression in HCT-116 spheroids determined from (A) after 144-h siRNA treatment. Each lane is representative of an independent pool of spheroids (N=3). (C) Immunocytochemistry images of serially sectioned HCT-116 spheroids after 144-h siRNA treatment with an Accell PPIB Control Pool or NTC Pool. Spheroids were then collected, fixed in 2% paraformaldehyde, embedded in optimal cutting (O.C.T.) temperature compound, flash frozen, and serially sectioned. Material was incubated with 1:200 dilution of primary antibody against PPIB (anti-cyclophilin B, Abcam ab16045) and secondary incubation with 1:500 dilution of Alexa 488-conjugated Goat-anti-rabbit antibody (Invitrogen A11034) and 1:1000 dilution of Hoechst 33342 (Molecular Probes H-3570). Scale bar = 200 µM.

 

---

 

Accell control siRNA demonstrates effective uptake and silencing in organotypic brain slices

Accell siRNA shows increased uptake with extended incubation. 250 µM cerebellar sections were prepared, cultured, and incubated for 3 hours (A) and 72 hours (B) with Accell Red Non-targeting control (NTC) siRNA (Cat# D-001960-01-05) before inspection by microscopy.

 

---

 

Robust gene target knockdown in primary lung fibroblasts using Accell 2.0 siRNA

 

IMR-90 human lung fibroblasts (5,000 cells / well) were plated in 96-well plates in IMR-90 media (EMEM supplemented with 10% FBS, L-glutamine and 0.1 mM non-essential amino acids). After adhesion and outgrowth for 16-18 hours, IMR-90 cells were at 80% confluency and experiments were initiated. After media removal, cells were washed with D-PBS and immersed in 100 mL Accell media with 1 µM Accell 2.0 siRNA. After 5 hours of incubation, 100 mL of IMR-90 media with 4% FBS was added to each well to result in 2% FBS. After culturing for 72 hours, total RNA was extracted and used for downstream RT-qPCR analysis of target mRNA abundance from PTEN, PPP2R1B, AKT1, and AKT2 genes relative to Non-targeting Control sample (NTC).