For these experiments, it is imperative to optimize transfection conditions to deliver efficiently nucleic acid molecules of interest, while maintaining cell viability. 1A). The 300 l siRNA/lipid complexes were added to freshly plated 5 105 cells in a 6-well plate. Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. This specific voltage and capacitance were selected after testing different electroporation parameters to achieve 50% cell survival 24 h post-electroporation. Preparation of Exosomes for siRNA Delivery to Cancer Cells This transfection methodology results in 83% DDX5 knockdown and 75% hnRNPA1 knockdown efficiencies at the protein level, as measured by immunoblot (Fig. However, the experimental conditions of RNAi-knockdown experiments are dependent on cell type, and gene expression levels need to be optimized correspondingly. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Lonza has developed five different Nucleofector Solutions designed to work for different cell lines/types. Transfer cells to 15 mL conical tubes (1 10, Resuspend the cells carefully in 100 L room temperature Nucleofector Solution V per sample. Tenenbaum SA, Lager PJ, Carson CC, Keene JD. Cells (1 106) were used/transfection on a 6-well plate with 250 nM siRNA duplex. The roles and targets transcripts of hnRNP A1 and the p68/DDX5 RNA helicase in pre-mRNA splicing can be studied by comparing alternative splicing patterns by RNA-seq after RNAi knockdown of these factors. Co-transfection is performed when the user wants to introduce both siRNA and a plasmid for expressing a protein into a cell. 2. Note 5). Western blotting is the most widely used technique for detecting proteins (see Although some differences in values for the isoforms tested for each of the specific target genes were observed, the effects of hnRNP A1 knockdown on different targets were generally conserved across different cell lines tested (data not shown). These data demonstrate that the transfection methods we developed result in an efficient knockdown of several genes of interest and resulted in changes in the pattern of several known hnRNP A1 splicing target pre-mRNAs to be detected by RT-PCR. For transfection of GM12878 cells, 4 106 cells/reaction (DDX5 knockdown) or 6 106 cells/reaction (knockdown of highly abundant proteins, e.g., hnRNP A1) were used. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. The authors also thank George Ghanim and Malik Francis for critical review of the manuscript. Gently mix and transfer the solution to the pre-incubated 6-well plate. Optimal siRNA Transfection Using an Electroporator Therefore, adding antibiotics to the transfection medium is not recommended. Electroporation gene therapy, or gene electrotransfer, has evolved greatly over the last few decades as a result of the remarkable progress in genetic sequencing, gene array analysis, gene cloning, gene expression detection, DNA manufacture and discovery and synthesis of siRNA. PDF Neon Transfection System - Thermo Fisher Scientific Keep in mind that while too much siRNA may lead to off-target or cytotoxic effects, too little siRNA may not reduce target gene expression effectively. Choosing the optimal transfection reagents for the cell type of interest may necessitate comparing reagents from different vendors. Electroporation Protocols: Preclinical and Clinical Gene Medicine . Alternatively, siRNAs can be endogenously expressed in the form of short hairpin RNA (shRNA), delivered to cells via plasmids or viral/bacterial vectors [6]. B) RT-PCR analysis of several pre-mRNA splicing target mRNAs reveals changes in hnRNP A1-dependent, alternative splicing events, recapitulated by RNAi in GM12878 cells. Delgado-Caedo A, Santos DG, Chies JA, Kvitko K, Nardi NB. Therefore, we recommend using standard purity siRNAs that are greater than 80% full length. Select the Nucleofector Program T-020 (see. Their delivery efficiency varies and can be cell line-dependent. Comparative analysis of metazoan chromatin organization. However, as a result of limitations in transfection efficiency, no RNAi-mediated mRNA and protein knockdown studies have been reported by use of the GM12878 cell line. When cell density is too low, cultures can become unstable. A) GM12878 cells (2 106) were transfected with GFP-expressing plasmid by use of Lonza Kit V with the indicated channels. GM12878 cells were maintained at a cell density of 3.5 105 cells/ml before transfection. Additionally, gene characterization via transgenic shRNA knockdown becomes difficult when one is targeting essential proteins, as this procedure may select for stably transformed cell lines that yield low mRNA and protein knockdown within the isolated cell clones. GM12878 cells were electroporated with nonspecific control siRNAs (scr si) or hnRNP A1 duplex siRNA duplexes. Incubate the solution at 90 C for 2 min, and then slowly cool to room temperature by placing the tube in a large beaker containing room temperature water for about 1 h. Briefly centrifuge the tube to bring down all droplets from the sides and lid of the tube. However, electroporation can induce high cell mortality, and often requires careful optimization of electroporation parameters (voltage, electric pulse length, and pulse number) to achieve high efficiency and low cell mortality. Small interfering RNA (siRNA) electroporation is a method that combines the use of siRNA molecules with electroporation to achieve targeted gene silencing in cells. The diagram below depicts an RNAi experiment workflow following siRNA design and synthesis. Electroporation of siRNA in vivo and in vitro represent highly efficient siRNA delivery method. For each T25 flask to be transfected, prepare 250 L of transfection reagent solution in a 1.5 mL Eppendorf tube by adding7.5 L of siLenFect to 242.5 L of serum-free medium (, For each T25 flask to be transfected, prepare 120 nM siRNA solution in 250 L serum-free medium in a 1.5 mL Eppendorf tube (, Add the siRNA solution to the diluted siLenFect solution (, Add 500 L of the siRNA/siLenfect complexes to the cells. and transmitted securely. Depending on the surface area of the vessel, the amount of reagents may need to be scaled up or down. Cells (2 106) were transfected with 2 g GFP-expressing plasmid in a 6-well plate. The publisher's final edited version of this article is available at, Negative control (scrambled or non-targeting) siRNA oligonucleotides. Efficiency: Electroporation is a highly efficient method for introducing siRNAs into cells, often with higher transfection rates than other non-viral methods, such as lipofection or chemical-based transfection. Various Sequences groups have developed specific guidelines for designing siRNAs [, Several siRNA manufacturers such as Thermo Fisher Scientific, QIAGEN, and GE Dharmacon have predesigned (and sometimes validated) siRNA sequences for most known genes in the human genome. siRNAs must be free of reagents carried over from synthesis, such as ethanol, salts, and proteins. The .gov means its official. The .gov means its official. The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. Visualize the RNA by staining with ethidium bromide, and verify that it is the expected size and intensity. Transient electroporation of chemically synthesized, preprocessed, short siRNA duplexes directly into the cell lines of interest may be faster and lead to more efficient mRNA and protein knockdown, especially for hard-to-transfect cell lines. However, because transfection reagents increase cell permeability, the delivery of antibiotics may also be increased, which could result in increased cytotoxicity. Sequence and chromatin determinants of cell-type-specific transcription factor binding. The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. To anneal single-stranded RNA, resuspend the lyophilized siRNA powder received from the vendor in RNase-free water at a final concentration of 100 M. Electroporation of siRNA to Silence Gene Expression in Here I describe the general protocol for using the Amaxa Nucleofector device and kit to deliver siRNA, using MIA PaCa-2 cells as an example. et al. There are multiple methods to deliver nucleic acid materials into a desired human cell line, from chemical transfection (e.g., cationic liposome-mediated transfection or calcium phosphate) and nonchemical transfection (e.g., electroporation). The cells were harvested for protein immunoblot analysis or RNA isolation, 72 h postsecond siRNA transfection. After a second round of siRNA transfection, the cells were harvested for preparation of protein lysates, which were subjected to immunoblotting with anti-hnRNP A1 or anti-DDX5 antibodies to assess the efficiency of siRNA-mediated protein knockdown. Depending on the known or predicted functions of the target gene, a variety of assays (cell growth and survival, migration, apoptosis, effects on downstream signaling, etc.) The burst of current is administered by an instrument known as an electroporator, which has the effect of opening up small holes in the cell's membrane, thereby enabling the siRNA molecule to enter the cell. An integrated encyclopedia of DNA elements in the human genome. For MIA PaCa-2, we use RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (see HEK293 cells were subcultured 24 h before transfection. Nonspecific control siRNA duplexes 1 [scrambled siRNA (scr si)] were purchased from Life Technologies (4390843). 3.3 Electroporation of siRNAs into Human Monocytes and Dendritic Cells. Aliquot the solution into new tubes in small volumes (e.g., 20 L) and store at20 C, if not to be used immediately. K562 is an immortalized myelogenous leukemia cell line. hnRNP A1 belongs to the A/B subfamily of ubiquitously expressed hnRNPs and has been shown to play a role in splicing/regulation of alternative splicing. Again, this demonstrates that electroporation is a more efficient way of delivering nucleic acids into hard to transfect cells or to deplete the activity of very highly expressed genes. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA .