Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

Poly-2'-DNP-RNAs with enhanced efficacy for inhibiting cancer cell growth

It is often believed that small interfering RNA (siRNA) is at least 10-fold more effective than the single-stranded antisense oligonucleotide for silencing the same target gene in the same cells. In view of the recent discovery that the RNA-induced silencing complex (RISC) contains only a single-stranded RNA (ssRNA) molecule and can be reconstituted using single-stranded antisense RNA, such a large difference in efficacy seems puzzling. One possible reason is that hybridization protects siRNA from hydrolysis by endogenous RNase activity until it is incorporated in the RISC, whereas ssRNA is rapidly hydrolyzed. Because the single-stranded poly-2'-O-(2,4-dinitrophenyl)-RNA (DNP-ssRNA) is both RNase resistant and membrane permeable, we synthesized homologous native siRNAs, DNP-siRNAs, native ssRNAs, and DNP-ssRNAs and made a comparative study of their efficacies for inhibiting the growth of two cancer cell lines with different overexpressed target genes under equivalent experimental conditions. It was found that the efficacy of antisense DNP-ssRNA is higher than that of the corresponding siRNA and that the efficacy of native siRNA for inhibiting cell growth can also be enhanced from 2-fold to 6-fold by replacing the native strands of RNA in siRNA with homologous DNP-RNA. Thermal denaturation data show that the hybridization affinity of the DNP-RNA/RNA duplex is higher than that of the native RNA/RNA duplex. Western blotting analysis of A549 cells treated with antisense DNP-ssRNAs containing single mismatching bases shows that the gene silencing by antisense DNP-ssRNA is as sequence specific as that by siRNA. The observed large enhancement of inhibition efficacy of native RNAs by DNP derivatization should be advantageous for both gene silencing studies and therapeutic applications.     

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.     

Ultrasound: mechanical gene transfer into plant cells by sonoporation

Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues.     

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.     

The role of bestrophin in airway epithelial ion transport

The purpose of this study was to identify Cl- channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl- channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT-PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl- flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl- channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.     

Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcεRI-activated RBL-2H3 basophilic cells

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcεRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcεRI-mediated signal transduction, degranulation and TNFα secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcεRI-mediated signal transduction, degranulation and TNFα secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.     

Cellular Delivery of siRNA Mediated by Fusion-Active Virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes (“virosomes”), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

Cellular delivery of siRNA mediated by fusion-active virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes ("virosomes"), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.     

Current clinical trials with non-coding RNA-based therapeutics in malignant diseases: A systematic review

This systematic review aimed to shed light on the trend of current clinical trials of non-coding RNA (ncRNA)-based therapeutics for malignant diseases. We conducted a database search for published literature and ongoing clinical trials using PubMed, clinicaltrials.gov, and University Medical Information Network (UMIN) clinical trial registry. To ensure that our review was based on up-to-date clinical trials, we limited our search to literature published within the last five years (January 2017–September 2022). Furthermore, due to the “clinical” nature of our review, we focused only on studies involving human participants. Among ncRNAs, microRNAs have been extensively explored in observational studies of malignant diseases as potential diagnostic markers and prognostic predictors, as well as for their therapeutic monitoring and profiling capabilities. As therapeutic agents, microRNA or siRNA were estimated in interventional human clinical trials and showed promising outcomes; however, the number of trials was small. Evidence and ongoing clinical trials in which ncRNAs other than microRNA or siRNA have been evaluated for their potential as therapeutic agents are limited. Here, we summarized microRNA as a potential therapeutic agent in malignant diseases, but most of the current evidence suggests that it is useful as a potential biomarker. siRNA is also a promising ncRNA technique in cancer, however more data from clinical trials are warranted for clinical use.     

Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization

Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [14C]-L-serine (a precursor of all sphingolipids). SMS activity and [14C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [14C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.     

肿瘤的超声波治疗

文中就超声波热疗,高强度聚焦超声以及低频超声波在肿瘤治疗中的应用进行了描述。早期主要是利用超声波的热效应来治疗肿瘤,近年来兴起的高强度聚焦超声是热疗法的另一发展。它瞬间使肿瘤组织温度升至65℃以上,导致靶区组织凝固和坏死来达到对肿瘤的“热切除”,是一种安全、有效的肿瘤治疗手段,具有无限潜力。尽管低频超声治疗肿瘤的机制尚不明了,但因其可以诱导细胞凋亡,为肿瘤的治疗提供了新的途径,其治疗作用已受到重视。     

面向经颅聚焦超声的多阵元相控阵关键技术

经颅聚焦超声是一种有效的神经调控技术，具有非侵入性、聚焦靶点多和焦点可调控等优势。但由于颅骨的强声衰减和非均质特性，聚焦超声经颅后存在焦点偏移、焦域能量不足以及颅骨烫伤等问题。多阵元超声相控阵可以修正超声经颅后的相位偏差和幅值衰减，实现准确、有效的颅内聚焦。本文首先介绍了换能器的阵元排布方式，进一步归纳了相控阵激励信号的调控方法，最后对其基础研究和临床应用进行了回顾与展望。     

IQ-domain GTPase-activating protein 1 regulates beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and adenomatous polyposis coli

Beta-catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that beta-catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of beta-catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of beta-catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased beta-catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of beta-catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that beta-catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The beta-catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of beta-catenin. In addition, beta-catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of beta-catenin function. We propose that in motile cells beta-catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein beta-catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.     

NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.     

Optimization procedure for small interfering RNA transfection in a 384-well format

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns.