Angela Reynolds | Polk County Public Schools (original) (raw)
Papers by Angela Reynolds
Small interfering RNA (siRNA) is a powerful functional genomic tool used to silence the expressio... more Small interfering RNA (siRNA) is a powerful functional genomic tool used to silence the expression of targeted genes. A genome-wide view of this phenomenon has allowed identification (in addition to specific gene knockdown) of both sequencespecific "off-target" effects (Jackson, 2003) and sequence non-specific toxicity affects . In this study we have used Agilent's in situ synthesized 60-mer oligonucleotide microarrays to systematically study siRNA-based off-targeting and toxic effects siRNAs are most often introduced into cells using lipid-mediated transfection. The large-scale expression signature represents a combination of both transfection and siRNA-specific effects. We have evaluated the degree of the cellular response due to siRNAbased effects and transfection-based artifacts. A previously reported siRNA-based induction of an interferon response is here observed only in the background of an invasive or toxic transfection protocol and can be sequence-dependent. Optimization of transfection conditions (minimization of lipid-based toxicity) or use of alternative delivery technologies can minimize siRNA-based toxicity. In conclusion, global expression effects of a given siRNA are minimal and may be controlled by careful sequence selection and experimental design.
Nature Cell Biology, 2008
To screen for motility in high throughput, we developed a robotic-driven pin to deliver a precise... more To screen for motility in high throughput, we developed a robotic-driven pin to deliver a precise scratch in confluent cell monolayers. The assay conditions were established using siRNAs targeting RHOA and RAC1 (refs 8, 9) and wound healing was evaluated after 12 h when the mocktransfected cells, migrating collectively as an epithelial cell sheet, close the wound by 50-60% ( ). The extent of motility, termed the 'area score' , was numerically quantified and a visual evaluation was also performed to provide a secondary
Methods in Enzymology, 2005
RNA interference is widely recognized for its utility as a functional genomics tool. In the absen... more RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.
Rna-a Publication of The Rna Society, 2006
Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi... more Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell typespecific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.
Cell, 2003
Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated in... more Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated into a ribo-1 Amgen, Inc. One Amgen Center Drive nucleoprotein complex that includes at least two members of the RISC, further linking siRNA and miRNA pro-Thousand Oaks, California 91320 2 Dharmacon, Inc. cessing (Mourelatos et al., 2002). Unlike siRNAs, which regulate mRNA levels through a cleavage event, miRNAs 1376 Miners Drive, #101 Lafayette, Colorado 80026 function by attenuating translation (Hutvá gner and Zamore, 2002). However, it was recently reported that a well-characterized let-7 miRNA, which naturally directs translational attenuation, can also cleave mRNA so long Summary as it is composed of a sequence that is completely complementary to the target (Hutvá gner and Zamore, Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer 2002). The finding that an miRNA can function as an siRNA (Doench et al., 2003; Zeng et al., 2003) indicates and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statis-that the degree of sequence identity between the regulatory RNA and its target may be the key factor in de-tical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor termining which form of posttranscriptional gene silencing is exploited. hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5-anti-sense (AS) terminal Several studies have shown that duplex unwinding is critical for the processing of both dsRNA and pre-miRNA base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal hairpin precursors (Bernstein et al., 2001; Nicholson and Nicholson, 2002) and is necessary for the formation of stability (⌬0.5 kcal/mol) at the 5-AS end than nonfunctional duplexes. Average internal stability of siRNA silencing-competent (activated) RISC (RISC*), thus highlighting the importance of helicase activity in the RNAi molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic process (Nykanen et al., 2001). In addition, recent studies in D. melanogaster cell lysates suggest that sequence thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA asymmetry and strand instability in the pre-miRNA hairpin precursor or siRNA duplex may contribute to selec-play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved tive strand processing and entry into RISC* (Schwarz et al., 2003 [this issue of Cell]). Taken together, these stud-in duplex unwinding and strand retention by RISC.
Nature Biotechnology, 2004
Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated proces... more Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi) 1-4 . RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi 5,6 . To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
Cell, 2003
Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated in... more Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated into a ribo-1 Amgen, Inc. One Amgen Center Drive nucleoprotein complex that includes at least two members of the RISC, further linking siRNA and miRNA pro-Thousand Oaks, California 91320 2 Dharmacon, Inc. cessing (Mourelatos et al., 2002). Unlike siRNAs, which regulate mRNA levels through a cleavage event, miRNAs 1376 Miners Drive, #101 Lafayette, Colorado 80026 function by attenuating translation (Hutvá gner and Zamore, 2002). However, it was recently reported that a well-characterized let-7 miRNA, which naturally directs translational attenuation, can also cleave mRNA so long Summary as it is composed of a sequence that is completely complementary to the target (Hutvá gner and Zamore, Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer 2002). The finding that an miRNA can function as an siRNA (Doench et al., 2003; Zeng et al., 2003) indicates and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statis-that the degree of sequence identity between the regulatory RNA and its target may be the key factor in de-tical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor termining which form of posttranscriptional gene silencing is exploited. hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5-anti-sense (AS) terminal Several studies have shown that duplex unwinding is critical for the processing of both dsRNA and pre-miRNA base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal hairpin precursors (Bernstein et al., 2001; Nicholson and Nicholson, 2002) and is necessary for the formation of stability (⌬0.5 kcal/mol) at the 5-AS end than nonfunctional duplexes. Average internal stability of siRNA silencing-competent (activated) RISC (RISC*), thus highlighting the importance of helicase activity in the RNAi molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic process (Nykanen et al., 2001). In addition, recent studies in D. melanogaster cell lysates suggest that sequence thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA asymmetry and strand instability in the pre-miRNA hairpin precursor or siRNA duplex may contribute to selec-play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved tive strand processing and entry into RISC* (Schwarz et al., 2003 [this issue of Cell]). Taken together, these stud-in duplex unwinding and strand retention by RISC.
Nature Methods, 2006
Off-target gene silencing can present a notable challenge in the interpretation of data from larg... more Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3¢ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.
Rna-a Publication of The Rna Society, 2006
Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duple... more Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.
Nature Protocols, 2007
Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understa... more Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.
Small interfering RNA (siRNA) is a powerful functional genomic tool used to silence the expressio... more Small interfering RNA (siRNA) is a powerful functional genomic tool used to silence the expression of targeted genes. A genome-wide view of this phenomenon has allowed identification (in addition to specific gene knockdown) of both sequencespecific "off-target" effects (Jackson, 2003) and sequence non-specific toxicity affects . In this study we have used Agilent's in situ synthesized 60-mer oligonucleotide microarrays to systematically study siRNA-based off-targeting and toxic effects siRNAs are most often introduced into cells using lipid-mediated transfection. The large-scale expression signature represents a combination of both transfection and siRNA-specific effects. We have evaluated the degree of the cellular response due to siRNAbased effects and transfection-based artifacts. A previously reported siRNA-based induction of an interferon response is here observed only in the background of an invasive or toxic transfection protocol and can be sequence-dependent. Optimization of transfection conditions (minimization of lipid-based toxicity) or use of alternative delivery technologies can minimize siRNA-based toxicity. In conclusion, global expression effects of a given siRNA are minimal and may be controlled by careful sequence selection and experimental design.
Nature Cell Biology, 2008
To screen for motility in high throughput, we developed a robotic-driven pin to deliver a precise... more To screen for motility in high throughput, we developed a robotic-driven pin to deliver a precise scratch in confluent cell monolayers. The assay conditions were established using siRNAs targeting RHOA and RAC1 (refs 8, 9) and wound healing was evaluated after 12 h when the mocktransfected cells, migrating collectively as an epithelial cell sheet, close the wound by 50-60% ( ). The extent of motility, termed the 'area score' , was numerically quantified and a visual evaluation was also performed to provide a secondary
Methods in Enzymology, 2005
RNA interference is widely recognized for its utility as a functional genomics tool. In the absen... more RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.
Rna-a Publication of The Rna Society, 2006
Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi... more Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell typespecific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.
Cell, 2003
Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated in... more Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated into a ribo-1 Amgen, Inc. One Amgen Center Drive nucleoprotein complex that includes at least two members of the RISC, further linking siRNA and miRNA pro-Thousand Oaks, California 91320 2 Dharmacon, Inc. cessing (Mourelatos et al., 2002). Unlike siRNAs, which regulate mRNA levels through a cleavage event, miRNAs 1376 Miners Drive, #101 Lafayette, Colorado 80026 function by attenuating translation (Hutvá gner and Zamore, 2002). However, it was recently reported that a well-characterized let-7 miRNA, which naturally directs translational attenuation, can also cleave mRNA so long Summary as it is composed of a sequence that is completely complementary to the target (Hutvá gner and Zamore, Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer 2002). The finding that an miRNA can function as an siRNA (Doench et al., 2003; Zeng et al., 2003) indicates and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statis-that the degree of sequence identity between the regulatory RNA and its target may be the key factor in de-tical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor termining which form of posttranscriptional gene silencing is exploited. hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5-anti-sense (AS) terminal Several studies have shown that duplex unwinding is critical for the processing of both dsRNA and pre-miRNA base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal hairpin precursors (Bernstein et al., 2001; Nicholson and Nicholson, 2002) and is necessary for the formation of stability (⌬0.5 kcal/mol) at the 5-AS end than nonfunctional duplexes. Average internal stability of siRNA silencing-competent (activated) RISC (RISC*), thus highlighting the importance of helicase activity in the RNAi molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic process (Nykanen et al., 2001). In addition, recent studies in D. melanogaster cell lysates suggest that sequence thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA asymmetry and strand instability in the pre-miRNA hairpin precursor or siRNA duplex may contribute to selec-play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved tive strand processing and entry into RISC* (Schwarz et al., 2003 [this issue of Cell]). Taken together, these stud-in duplex unwinding and strand retention by RISC.
Nature Biotechnology, 2004
Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated proces... more Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi) 1-4 . RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi 5,6 . To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
Cell, 2003
Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated in... more Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Mature miRNAs are incorporated into a ribo-1 Amgen, Inc. One Amgen Center Drive nucleoprotein complex that includes at least two members of the RISC, further linking siRNA and miRNA pro-Thousand Oaks, California 91320 2 Dharmacon, Inc. cessing (Mourelatos et al., 2002). Unlike siRNAs, which regulate mRNA levels through a cleavage event, miRNAs 1376 Miners Drive, #101 Lafayette, Colorado 80026 function by attenuating translation (Hutvá gner and Zamore, 2002). However, it was recently reported that a well-characterized let-7 miRNA, which naturally directs translational attenuation, can also cleave mRNA so long Summary as it is composed of a sequence that is completely complementary to the target (Hutvá gner and Zamore, Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer 2002). The finding that an miRNA can function as an siRNA (Doench et al., 2003; Zeng et al., 2003) indicates and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statis-that the degree of sequence identity between the regulatory RNA and its target may be the key factor in de-tical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor termining which form of posttranscriptional gene silencing is exploited. hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5-anti-sense (AS) terminal Several studies have shown that duplex unwinding is critical for the processing of both dsRNA and pre-miRNA base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal hairpin precursors (Bernstein et al., 2001; Nicholson and Nicholson, 2002) and is necessary for the formation of stability (⌬0.5 kcal/mol) at the 5-AS end than nonfunctional duplexes. Average internal stability of siRNA silencing-competent (activated) RISC (RISC*), thus highlighting the importance of helicase activity in the RNAi molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic process (Nykanen et al., 2001). In addition, recent studies in D. melanogaster cell lysates suggest that sequence thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA asymmetry and strand instability in the pre-miRNA hairpin precursor or siRNA duplex may contribute to selec-play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved tive strand processing and entry into RISC* (Schwarz et al., 2003 [this issue of Cell]). Taken together, these stud-in duplex unwinding and strand retention by RISC.
Nature Methods, 2006
Off-target gene silencing can present a notable challenge in the interpretation of data from larg... more Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3¢ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.
Rna-a Publication of The Rna Society, 2006
Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duple... more Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.
Nature Protocols, 2007
Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understa... more Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.