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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. 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. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. 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. et al. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. For instance, RNA-sequencing (seq) experiments often use RNAi technologies to knock down mRNA expression and protein levels of the gene of interest to study gene regulation and mRNA isoform expression. IV. With the ENCODE project aiming to identify all functional elements encoded in the human genome, the project has generated and made publicly available vast amounts of high-throughput sequencing data for transcription profiles, chromatin marks, and transcription factors in ChIP-seq.1220 Both GM12878 and K562 cells possess a relatively normal karyotype, unlike HEK293 and HeLa cells that have undergone extensive chromosomal aneuploidy. Here, we present an efficient transfection method that can be used for mRNA and protein knockdown studies in ENCODE Tier 1 cell lines GM12878 and K562 for gene expression, mRNA splicing isoform pattern, and functional studies. Master mix can be prepared if replicates of the same siRNA concentrations are being carried out. siRNAs must be free of reagents carried over from synthesis, such as ethanol, salts, and proteins. Introduction to Transfection. In recent years, a number of commercial manufacturers have started to offer siRNA oligonucleotide synthesis, which has greatly facilitated the use of synthetic siRNAs in research. 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. 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. Again, 2 g GFP-expressing plasmid was transfected into 2 106 cells and harvested 24 h post-transfection. 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). When performing an RNAi experiment, make sure that you have the following on hand: Figure 1: RNAI workflow following siRNA design and synthesis. One major difference between the two methods is that RNA can only be transiently transfected. Electroporation has so far given us the best results, but even so we've only gotten about a 20% transfection efficiency. 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 . 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. et al. Small interfering RNA (siRNA) electroporation is a method that combines the use of siRNA molecules with electroporation to achieve targeted gene silencing in cells. Not for use in diagnostic procedures. We've made it easier for you to quickly find and buy products for every step in your transfection workflow. . One major difference between the two methods is that RNA can only be transiently transfected. Aliquot the resuspended/annealed siRNA into new tubes and store at 20 C. Twenty-four hours post-transfection, the cells were stained with Hoechst nuclear DNA stain (1 g/ml) for 1 h. The percentage of GFP-expressing cells was quantified by use of the ImageJ software program (NIH). siRNA is used for RNAi studies that examine the effects of gene knockdown. The human GM12878 lymphoblastoid cell line is used extensively by the ENCODE Project and is particularly attractive because of the vast amount of sequencing data, including multiple RNA immunoprecipitation (RIP)-chromatin immunoprecipitation (ChIP), RIP-seq, and ChIP-seq experiments, readily available.1220 Yet, RNAi studies have not been routinely performed in this line as a result of technical difficulties with nucleic acid delivery by cationic lipid-mediated transfection. 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). The optimal amount of siRNA and its capacity for gene silencing are influenced in part by properties of the target gene products, including the following: mRNA localization, stability, abundance, as well as target protein stability and abundance. 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. MicroRNA modulation of RNA-binding protein regulatory elements. siRNA-mediated gene silencing in vitro and in vivo. This method has advantages over shRNA-mediated knockdown, as it does not require generation of stable cell lines and bypasses use of endogenous microRNA processing pathways, while providing higher effective concentrations of siRNA delivered to the cell. This work was supported by NIH/NCI grants CA169281 and CA191923 to H.H. Baroni TE, Chittur SV, George AD, Tenenbaum SA. Here, we report a transfection protocol by use of electroporation of siRNAs that results in efficient and reproducible RNAi knockdown of genes of interest in human GM12878 cells. The major variables that impact siRNA transfection efficiency are the following: It is important to include a positive control in each experiment. The company has developed an extensive database of cell type-specific electroporation programs and solutions, which has minimized the optimization process for end users. Also, dsRNA contaminants longer than 30 bp are known to alter gene expression by activating the nonspecific interferon response and causing cytotoxicity (Stark et al., 1998). Here, we report a transfection protocol that uses electroporation of siRNAs, resulting in efficient and reproducible RNAi knockdown of genes of interest in human GM12878 cells. This method will pave the way for the study of hypotheses generated regarding the function of genes and proteins that result from the examination of the vast amount of ENCODE Project data readily available and that will be generated in the future with the use of GM12878 and K562 cells. Although reduction in transcript expression usually results in Western Blotting decreased protein abundance, mRNA levels do not always correlate with protein levels. 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. siRNA electroporation has several advantages over other gene silencing methods: siRNA electroporation has several applications in molecular biology and biomedical research, including: Despite its advantages, siRNA electroporation also has some limitations, such as potential off-target effects due to the unintended silencing of genes with similar sequences, and challenges associated with in vivo delivery of siRNAs to specific tissues or organs. 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. Potential challenges and problems associated with the siRNA technology are also discussed. To further optimize the transfection in GM12878 cells, different programs, including X-002, X-005, Y-001, and Y-005, with similar electric-field strengths and pulse durations to that of the X-001 programs were then tested (Fig. This work was supported by the U.S. National Institutes of Health Grants P50 GM102706 (to the Center of RNA Systems Biology, J. Cate, PI) and GM097352 (to D.C.R.). Culture medium (1.5 ml) was divided in aliquots into each well in a 6-well plate and equilibrated to 37C in the incubator. Cells (2 106) were transfected with 2 g GFP-expressing plasmid in a 6-well plate. Electroporation of siRNA in vivo and in vitro represent highly efficient siRNA delivery method. 8600 Rockville Pike siPORT siRNA Electroporation Buffer I. II. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA. However, for hard-to-transfect human cell lines, such as embryonic stem cells, lymphoid, or other lines, electroporation-mediated transfection yields higher transfection efficiencies. The transfection mix was incubated at room temperature for 5 min. Further optimization of electroporation of GM12878 cell transfection efficiency by use of GFP-expressing plasmids and immunoblotting. Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. PCR products were resolved, visualized, and quantitated by use of an Agilent Technologies (Santa Clara, CA, USA) Bioanalyzer. If you see maximal effect above/below a pre-determined threshold level with this control, you know that measurements from other experiments tested on the same day are reliable. 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 critical to maintain the cell density between 2 105 and 1 106 cells/ml for GM12878 cell viability. 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. Small interfering RNA (siRNA) electroporation is a method that combines the use of siRNA molecules with electroporation to achieve targeted gene silencing in cells. Transfection uses a lipid carrier to facilitate the cellular uptake of siRNA. Remove the cuvette from the holder once the program is finished. 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. RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. However, for some cell lines and cell types, particularly primary cells and suspension cells, these methods yield low efficiency. K562 is an immortalized myelogenous leukemia cell line. 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. (2008), AsiDesigner: exon-based siRNA design server considering alternative splicing, RNA isolation and real-time quantitative RT-PCR, Quantification of mRNA using competitive RT-PCR with standard-curve methodology, Analyzing real-time PCR data by the comparative C(T) method, Killing the messenger: short RNAs that silence gene expression, Gilmore IR, Fox SP, Hollins AJ, Akhtar S (2006), Delivery strategies for siRNA-mediated gene silencing, siRNAs: applications in functional genomics and potential as therapeutics, https://rnaidesigner.thermofisher.com/rnaiexpress, http://dharmacon.gelifesciences.com/design-center. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA . Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+ 500 uL of cells is mixed with 50-200 ug of DNA (or 5 uL siRNA) in a 0.4 cm cuvette. A number of lipid carriers (transfection reagents) specifically developed for siRNA oligonucleotides are commercially available. 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. The quality of siRNA can significantly influence RNAi experiments. 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. 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. The presence of the plasmid may decrease transfection efficiency of all cargo (plasmid and siRNA) when a lipid transfection reagent is used, making transfection optimizations very important. Optimization of electroporation of GM12878 cell transfection efficiency by use of GFP-expressing plasmids. Gene knockdown can be detected as early as 4 h and could last up to 5 days, and even 7 days in some cases [. Note 5). 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. In this chapter, I will focus on procedures that utilize commercially synthesized siRNAs to knockdown gene expression in mammalian cells. Mix the solution well by pipetting up and down a few times. Mix by rocking the flasks back and forth several times. The purpose of RNA transfection is similar to that of plasmid transfection. However, because transfection reagents increase cell permeability, the delivery of antibiotics may also be increased, which could result in increased cytotoxicity. Once knockdown of the siRNA-targeted gene is confirmed, assays can then be carried out to investigate resulting functional effects. Cells (1 106) were used/transfection on a 6-well plate with 250 nM siRNA duplex. The site is secure. 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). For this reason the GM12878 and K562 cell lines were chosen for use in the Tier 1 ENCODE Project, along with H1 human embryonic stem cells. The final concentration of the annealed siRNA duplex is 20 M. Twenty-four hours post-transfection, the medium was changed with fresh media. Co-transfection is performed when the user wants to introduce both siRNA and a plasmid for expressing a protein into a cell. Note 3) are siRNAs known to downregulate the expression of a specific gene. As a library, NLM provides access to scientific literature. The manufacturer has optimized programs for a number of cell lines. Complex formation between transfection agents and siRNA should be performed in reduced-serum or serum-free medium, so that serum components will not interfere with the reaction. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Transfection of RNA is an offshoot of classic transfection technologies for introducing RNA into cells. RNA interference: listening to the sound of silence. 1B). The diagram below depicts an RNAi experiment workflow following siRNA design and synthesis. After searching for other Lonza programs used for other human B cell lines, we also tested programs Z-001, P-016, and U-009, in addition to Y-001, which gave the highest GFP transfection efficiency in the initial testing. Expression data suggest that hnRNP A1 and DDX5 genes are highly expressed in human cells and are essential for proper RNA splicing and cell survival, making both hnRNP A1 and DDX5 potentially problematic candidates for RNAi-mediated mRNA and protein knockdown studies. An official website of the United States government. Inclusion in an NLM database does not imply endorsement of, or agreement with,

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