Copyright 2023 Electroporation Protocols, method that combines the use of siRNA molecules. et al. Transfection of THP-1 cells with plasmids or small interfering RNA (siRNA) is achieved by electroporation using the Lonza Nucleofector technology (Basel, Switzerland). To assess transfection efficiency, we recommend including the BLOCK-iT Fluorescent Oligo in every experiment. Twenty-four hours post-transfection, the cells were stained with Hoechst nuclear DNA stain (1 g/ml) for 1 h. All cells were imaged with an inverted fluorescence microscope (upper), and the percentage of GFP-expressing cells was quantitated (lower). If the cell line or type is not on the list with an optimized protocol, then an optimization kit should be obtained. Mix by rocking the flasks back and forth several times. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. RNA interference, RNAi, siRNA, Gene silencing, Transfection, Electroporation, Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE The authors also thank George Ghanim and Malik Francis for critical review of the manuscript. Specificity: siRNAs are designed to target specific mRNA sequences, allowing for the selective inhibition of individual genes. Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. For example, mRNA measurement can overestimate knockdown of genes whose protein products have long half-lives. Diluted siRNA duplexes were added to diluted Lipofectamine RNAiMAX reagent and incubated at room temperature for 15 min. Equivalent protein amounts of each sample were subjected to SDS-polyacrylamide and immunoblotting with the following antibodies: TurboGFP (PIPA522688; 1:1000 dilution; Thermo Fisher Scientific, Waltham, MA, USA), hnRNP A1 (4B10; 1:1000 dilution; Sigma, St. Louis, MO, USA), DDX5 (NB200-351; 1:1000 dilution; Novus Biologicals, Littleton, CO, USA), and -actin (AC-74; 1:3000 dilution; Sigma). The 300 l siRNA/lipid complexes were added to freshly plated 5 105 cells in a 6-well plate. The Neon device is preprogrammed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type, or you can program and store up to 50 cell-specific protocols in the Neon device database. Transfer cells to 15 mL conical tubes (1 10, Resuspend the cells carefully in 100 L room temperature Nucleofector Solution V per sample. Additional mRNA transcripts, including eIF4EBP3, ASPH, USPL1, and SYNCRIP, showed only very slightly spliced isoform changes. To optimize siRNA knockdown conditions, a cell line expressing relatively high levels of the target gene should be used. The procedures are based on the instruction manual provided by the manufacturer with some modifications. 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. Cellular protein lysates were prepared in lysis buffer [50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS] containing protease inhibitors and quantified by use of the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). siRNA-mediated knockdown of hnRNP A1 and DDX5 proteins in GM12878 and K562 cells. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. *P (P < 0.05), calculated by use of a 2-tailed Student's t test, statistically significant. Careers, Unable to load your collection due to an error. Electroporation also exhibits a more immediate knockdown effect, as it does not rely on cellular transcriptional machinery for expression. However, because transfection reagents increase cell permeability, the delivery of antibiotics may also be increased, which could result in increased cytotoxicity. Immediately after electroporation, 400 l of the pre-equilibrated culture medium to the cuvette was added and transferred to a 6-well plate. Nevertheless, siRNA electroporation remains a valuable tool in molecular biology and biomedical research for studying gene function and developing novel therapeutic strategies. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Before The authors thank Dr. James Manley (Columbia University) for providing the hnRNP A1 siRNA target sequence. Kundaje A, Kyriazopoulou-Panagiotopoulou S, Libbrecht M, et al. Here I describe the general procedures for performing these two methods (see Optimization of electroporation of GM12878 cell transfection efficiency by use of GFP-expressing plasmids. For siRNA delivery using electroporation, siRNA quantity has a less pronounced effect, but typically 1 g/50 L cells (1.5 M) of siRNA (range 0.52.5 g/50 L cells or 0.753.75 M) is sufficient. Uptake of the fluorescent oligomer by at least 80% of cells correlates with high efficiency. Close the cuvette with the cap. 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. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation and poly-ethylenimine (PEI) technologies. Workflow for optimization of electroporation conditions for leukemia cells . Cell culture medium appropriate for the cells being cultured. Transfection System offers open and transparent protocols that are optimized for ease of use and simplicity. Health of cultured cells. GFP protein expression levels were quantified from the immunoblots by use of Image Lab software (Bio-Rad Laboratories) and normalized against -actin, used as a loading control. Transfer cell/siRNA mixture into a certified cuvette (included in the Nucleofector kit). Optimized siRNA Delivery into Primary Immune Cells Using Electroporation | SpringerLink Home RNA Interference and CRISPR Technologies Protocol Optimized siRNA Delivery into Primary Immune Cells Using Electroporation Mouldy Sioud Protocol First Online: 01 February 2020 3078 Accesses 2 Citations For gene knockdown studies, one widely used strategy for RNAi knockdown studies makes use of a precursor miRNA (pre-miRNA) mimic encoded by a short hairpin (sh)RNA.36 This method uses a plasmid expression vector with an RNA polymerase III U6 small nuclear RNA promoter to drive expression of the shRNA, which is then processed in the cytoplasm to generate siRNAs that target their specific gene transcripts.710 Here, the siRNA exists in the cell, as long as the plasmid vector continues to be expressed, generally by selection of drug-resistant cell lines expressing the U6-shRNA-encoding gene. However, for some cell lines and cell types, particularly primary cells and suspension cells, these methods yield low efficiency. Knocking-down cyclin A(2) by siRNA suppresses apoptosis and switches differentiation pathways in K562 cells upon administration with doxorubicin. Use RNase-free reagents, tubes, and barrier pipette tips for preparing RNA for transfection. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Chemically synthesized siRNAs are relatively simple and quick to generate. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. 1, 2 Although a powerful technique, mRNA and protein knockdown efficiency is limited by the effective concentration of siRNA that can be introduced into the cytoplasm of . The same siRNA transfection protocol was used as in GM12878 cell electroporation-mediated transfection, with the exception of use of a Nucleofector program (T-016) for K562 cell transfection. . These short interfering RNAs are then incorporated into and direct the RNA-induced silencing complex (RISC) to the target RNA. The expressed shRNA must compete against endogenous microRNA processing pathways in the cell, thereby limiting the concentration of shRNA produced and the effective knockdown efficiency. RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. For the purposes of this study, electroporation channels and cell densities were optimized with the highest electroporation efficiencies by use of Lonza Program U-009 with 4 106 cells (Kit V) and Program Y-001 (Kit R). Visualize the RNA by staining with ethidium bromide, and verify that it is the expected size and intensity. The plus is that high siRNA transfections can be seen (70-95%), but the downside is that a lot of siRNA and cells have to be used. Total RNA (1 g) was reverse transcribed (170-8891; Bio-Rad Laboratories), according to the manufacturers instructions, and subjected to RT-PCR by use of the following temperature cycles: 98C for 30 s (1 cycle); 98C for 10 s, 60C for 30 s, and 72C for 30 s (35 cycles); and 72C for 5 min (1 cycle). Electroporation has so far given us the best results, but even so we've only gotten about a 20% transfection efficiency. Not for use in diagnostic procedures. Note 5). Splicing analysis that uses RT-PCR reactions was also performed from RNA isolated from the hnRNP A1 siRNA knockdown samples, and compared with the control samples from scr si-transfected samples. Yet, we did not observe an efficient knockdown of hnRNP A1 or DDX5 in GM12878 cells under this condition. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. [Effect of Gli1 gene silencing on proliferation of K562 cells and its mechanisms]. Gene-specific degradation of mRNA is one way to silence individual gene expression post-transcriptionally. A users guide to the encyclopedia of DNA elements (ENCODE). and transmitted securely. The negative control siRNAs can be siRNA sequences that have the same nucleotide composition as the gene-specific siRNA, but lack significant sequence homology to the human genome or siRNAs that have been designed to have no known homology to the human genome (often called non-targeting siRNA). Developed in 1998, siRNAs continue to be an easy and effective method for dramatically reducing the mRNA and protein products from a gene of interest within a chosen cell line.1, After a 2nd round of siRNA transfection, the cells were harvested for total RNA isolation and subjected to RT-PCR analysis. Select the Nucleofector Program T-020 (see. These parameters need to be modulated carefully to maximize the transfection efficiency while maintaining cell viability. We've made it easier for you to quickly find and buy products for every step in your transfection workflow. sharing sensitive information, make sure youre on a federal This is a ready-to-use trypsin solution containing 0.025% trypsin and 0.01% EDTA in PBS. Incubate the cells at 37 C in a humidified 5% CO. Assess knockdown efficiency or examine functional effects of the knockdown 2472 h following electroporation. Therefore, we recommend using standard purity siRNAs that are greater than 80% full length. The same protocol reported here can be used to examine the effects of RNAi depletion on cells after knockdowns of any other gene of interest. For those hard-to-transfect cells, electroporation-based methods are often used to deliver nucleic acids. RNAi can be achieved without much difficulty for easily transfectable cells lines, such as HEK293 and HeLa, by use of liposome-mediated transfection methods to deliver siRNAs to host cells. Note 4). These data indicated that the highest level of GFP expression in GM12878 cells was achieved by transfecting 4 106 cells by use of the U-009 program (Fig. Experimental results demonstrate that the Las1L ( = 0.27; P = 0.005) and eIF4EBP3 ( = 0.15; P = 0.001) pre-mRNAs are alternatively spliced as a result of 75% hnRNP A1 protein knockdown in GM12878 cells.Additional, pre-mRNA transcripts, including eIF4EBP3, ASPH, USPL1, and SYNCRIP, showed very slightly spliced mRNA isoform changes. It is recommended that a mock transfection with only siLenfect (no siRNA added) be included as a control. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. The concentration of siRNA needed for efficient knockdown may vary depending on cell lines used and the gene target itself. This can make shRNA-mediated RNAi knockdown an ineffective technique for highly expressed transcripts. Comparative analysis of metazoan chromatin organization. Introduction to Transfection. The overall transfection efficiency is influenced by the amount of transfection agent complexed to the siRNA. Tenenbaum SA, Lager PJ, Carson CC, Keene JD. Choosing the optimal transfection reagents for the cell type of interest may necessitate comparing reagents from different vendors. et al. Alternative pre-messenger RNA (pre-mRNA) splicing is one of the major mechanisms used by metazoans to regulate gene expression and to increase the functional diversity of the eukaryotic proteomes. The protein we're looking at arrests the cell cycle, so we need to create. However, if too many cells are used, and the amount of siRNA is not increased proportionally, the concentration of siRNA in the sample may be too low to effectively elicit gene silencing. The authors thank University of California Berkeley Biological Imaging Facility for the use of the fluorescence microscope and the facility's assistance. The optimal siRNA concentration may vary from cell line to cell line. In data not shown here, we also attempted to electroporate 4 106 GM12878 cells in the presence of 100 pmol siRNA by use of the Gene Pulser (220 V, 975 F; Bio-Rad Laboratories) for the knockdown studies. (a) To determine optimal electroporation conditions, incubate cells with nonspecific siRNA or siRNA targeted to a gene that is necessary for maintenance of the growth/viability of the cells of interest.In the absence of a gene target that is specific to the cell line of interest, siRNA targeting PLK1 can be used as a . In general, the manufacturers recommended procedures should be used as a starting point for optimization. A) GM12878 (left) and K562 cells (right) were transfected with nonspecific control siRNA oligos (scr si) or hnRNP A1 or DDX5 siRNA duplexes. 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.) GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. These volumes are for half of a final plate of cells. To optimize, titrate the transfection agent over a broad dilution range, and choose the most dilute concentration that still gives good gene knockdown. Dispense RMPI complete medium into 6-well tissue culture plate (2.5 mL/well) and incubate at 37C. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Positive control siRNAs are used to monitor the efficiency of siRNA delivery to cells. A number of lipid carriers (transfection reagents) specifically developed for siRNA oligonucleotides are commercially available. If you suspect that a preparation of siRNA may be degraded, check the integrity of the siRNA by running ~2.5 g on a non-denaturing 1520% acrylamide gel. Please refer to the manufacturers website for details (. Electroporation of siRNA in vivo and in vitro represent highly efficient siRNA delivery method. Here, the pancreatic cancer cell line MIA PaCa-2 is used as an example. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. 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. Two methods have been widely used to deliver chemically synthesized oligonucleotides into mammalian cells: transfection and electroporation. IV. (2007), A protocol for designing siRNAs with high functionality and specificity, Park YK, Park SM, Choi YC, Lee D, Won M The cell pellets were resuspended with the siRNA duplex suspension; then, cells/siRNA duplex oligo suspensions were transferred into cuvettes and electroporated. 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). It is advisable to first consult the literature to determine if any other groups have reported siRNA transfection in the same cell lines/types, and then start with the same transfection reagents and conditions. GM12878 cells were electroporated with nonspecific control siRNAs (scr si) or hnRNP A1 duplex siRNA duplexes. Electroporation is a technique that applies an electric field to cells, temporarily permeabilizing the cell membrane and allowing the uptake of molecules, such as siRNAs, that would otherwise not be able to cross the membrane. However, for hard-to-transfect human cell lines, such as embryonic stem cells, lymphoid, or other lines, electroporation-mediated transfection yields higher transfection efficiencies. mRNA is introduced into cells to express the encoded protein, and study gene function and regulation. Work areas should be wiped down with 70% ethanol or other RNase decontamination solution such as, BLOCK-iT Alexa Fluor Red Fluorescent Control, Silencer Select GAPDH Positive Control siRNAs. No culture medium addition or replacement is usually required following transfection, but changing the media can be beneficial in some cases, even when serum compatible reagents are used. Insert the cuvette containing the cell/siRNA solution into the Nucleofector Cuvette Holder. Add 1 mL of trypsin solution to the cells and incubate in a humidified CO. On the second day, 1560 min before transfection, aspirate medium from the flask and add 2.5 mL fresh serum-containing growth medium to the cells. In contrast to commonly used human cell lines, such as human embryonic kidney (HEK)293 and HeLa, the GM12878 and K562 cell lines have more stable chromosome karyotypes. Amaxas Nucleofector (Lonza Cologne AG) technology is an advanced electroporation technology that has been widely used for delivery of siRNA and other nucleic acids to hard-to-transfect cells. Forty-eight hours post-transfection, the cells were electroporated again with a second round of siRNAs. Electroporation involves jolting the cell with a quick burst of electrical current in the presence of the siRNA molecule. Transfection method Transfection conditions Quality and quantity of siRNA The goal of transfection optimization is to determine the conditions that will provide maximum gene knockdown while maintaining an acceptable level of viability for the particular cell type (see sidebar, Two-Step Optimization Protocol). 3B, where the scr si-transfected samples served as the control. This technique allows for the efficient nonviral delivery of plasmids, DNA, RNA, or siRNA into primary cells or cell lines even if the cells are not or are only slowly proliferating. hnRNP A1 and DDX5 siRNAs were electroporated into GM12878 cells in parallel with scr si as controls. For the cationic liposome-based transfection protocol, 5 105 cells were plated/reaction. Specific inhibition or knockdown of gene expression in cultured cells has been widely used to study the effects of loss-of-function mutation in individual genes. Not for use in diagnostic procedures. As a tool for knocking down the expression of individual genes post transcriptionally, RNAi has been widely used to study the cellular function of genes. 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. Non-targeting siRNAs are commercially available from a variety of vendors (e.g., QIAGEN or GE Dharmacon). These siRNAs serve as negative controls. 1. The following primers sets were used for RT-PCR validation: synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP) cDNA was amplified by use of forward: 5-CGATACCATCGGACAGGATT-3 and reverse: 5-TTGAAGAACTGCCAATGCAC-3 primers; zinc finger, CCHC domain-containing 17 (ZCCHC17) cDNA was amplified by use of forward: 5-AAGGCCTGAGACCATGGAA-3 and reverse: 5-GCCCCATAGTCTGTCACCAT-3 primers; neurofibromin 1 (NF1) cDNA was amplified by use of forward: 5-CGAAGTGTGTGCCACTGTTT-3 and reverse: 5-GGTGAGACAATGGCAGGATT-3 primers; ubiquitinspecific proteaselike 1 (USPL1) cDNA was amplified by use of forward: 5-ACGTTGCAACTAGGGTGGAG-3 and reverse: 5-CAAGCAGGGCAATACTCATCT-3 primers; eukaryotic translation initiation factor 4E-binding protein 3 (eIF4EBP3) cDNA was amplified by use of forward: 5-GGAGAACTCTCCTGCCCTTT-3 and reverse: 5-AGGATACACAGGAGGCATCG-3 primers; Las 1-like protein (Las1L) cDNA was amplified by use of forward: 5-TTGAACAGTTGGCAGCTTTG-3 and reverse: 5-AACTGAGAGAACGGCTTTGG-3 primers; and aspartyl/asparaginyl -hydroxylase (ASPH) cDNA was amplified by use of forward: 5-TCGAAGATGAAGCAAAAGAACA-3 and reverse: 5-CTTCCACGTGGTAACTATGCTC-3 primers. 22 such as Lipofectamine 2000 or RNAiMax (Invitrogen, Life Technologies), as well as electroporation.23 In contrast, numerous lipid-based transfection reagents and conditions tested in GM12878 cells were not successful at different cell densities (5 105, 1 1064 106 GM12878 cells) by use of 150 pmol DDX5 or hnRNP A1 siRNAs (data not shown). official website and that any information you provide is encrypted Transfection protocols vary from reagent to reagent and often require optimization for different cell types (and sometimes even different cell lines of the same type). Grow MIA PaCa-2 cells in a T75 cell culture flask to 7090% confluency. The degree of the response to a particular RNA or siRNA is directly linked to its transfection efficiency. Cells were then stained with Hoechst nuclear DNA stain, and the transfection efficiency was determined. K562 is an immortalized myelogenous leukemia cell line. siRNAs are short, double-stranded RNA molecules that can specifically bind to and degrade complementary mRNA molecules, thus inhibiting gene expression. Alternatively, siRNAs can be endogenously expressed in the form of short hairpin RNA (shRNA), delivered to cells via plasmids or viral/bacterial vectors [6]. Again, 2 g GFP-expressing plasmid was transfected into 2 106 cells and harvested 24 h post-transfection. et al. For lipid-mediated reverse transfections, 10 nM of siRNA (range 130 nM) is usually sufficient. The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. There are five basic modes of alternative splicing that include the following: alternative 5 splice site (alternative donor site), alternative 3 splice site (alternative acceptor site), mutually exclusive exons, exon skipping (cassette exon), and intron retention. Similar magnitudes of changes in these splicing events were observed in HEK293 and K562 cells after siRNA treatment (>90% protein knockdown efficiency). In 2006, Prechtel et al. Refer to the manufacturers manual for recommended medium volumes and the amount of reagents for different culture vessels. Images shown in A have been inverted for easier viewing. The manufacturer has also developed optimized protocols for a number of cell lines and primary cell types. 3A, right) and HEK293 cell lines (data not shown). Because siRNA oligonucleotides target mRNA for degradation, Gene Knockdown RT-PCR can be used to measure effects on gene expression using Using Reverse negative control siRNA-treated cells and gene-specific, siRNA-trea-Transcription ted cells. RISC is a nuclease complex that is responsible for the ultimate destruction of the target RNA and gene silencing [2]. Mix the solution well by pipetting up and down a few times. Electro-transfection is a physical gene transfer. HEK293 cells were subcultured 24 h before transfection. Kits containing the optimized Nucleofector Solution recommended by the manufacturer should be purchased.