CERS6 Human Pre-designed siRNA Set A

Cat. No.: C1466355

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Descripción general

CERS6 Human Pre-designed siRNA Set A contains three designed siRNAs for CERS6 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components CERS6 siRNA-1: 5 nmol (HPLC) CERS6 siRNA-2: 5 nmol (HPLC) CERS6 siRNA-3: 5 nmol (HPLC) siRNA Negative Control: 5 nmol (HPLC) FAM-labeled siRNA Negative Control: 5 nmol (HPLC) GAPDH siRNA Positive Control:5 nmol (HPLC)

Specifications

Condiciones de almacenamiento de almacenamiento

Store at -20°C

Enviado en

Ice chest + Ice pads

Este producto requiere envío en cadena de frío. Los servicios terrestres y otros servicios económicos no están disponibles.

Product Line

Productos biológicos

Application Protocols

Item-specific note

• No validated, item-specific protocols, sequences, or recommended dilutions are provided in the Product Data; consult the CoA/Spec Sheet if available.

General/literature protocol for siRNA knockdown (adherent cells; 24-well example)

1. Day −1: Seed 5×10^4 cells/well to achieve ~60% confluence at transfection.
2. Prepare solutions: Dilute siRNA to 20 nM in 50 µL serum-free medium. In a separate tube, dilute transfection reagent per manufacturer (e.g., 1–2 µL in 50 µL). Incubate 5 min.
3. Complexation: Combine the two solutions gently. Incubate 10–20 min at room temperature to form lipid–siRNA complexes.
4. Delivery: Add complexes dropwise to wells containing 400 µL complete medium (final siRNA ~10 nM). Swirl gently.
5. Incubation: Culture 24–72 h. If cytotoxicity is observed, replace medium after 4–6 h.
6. Assessment: Extract RNA at 24–48 h for qRT-PCR; prepare protein lysates at 48–96 h for Western blot of CERS6. Include non-targeting and positive control siRNA.
7. Lipidomics (optional): Extract lipids (Bligh–Dyer) and quantify ceramide species by LC–MS to confirm pathway impact.

Troubleshooting

• Low knockdown: Titrate dose, test alternative duplexes from the set, verify cell health, and confirm reagent activity with a positive control.
• High toxicity: Reduce lipid amount, shorten exposure, or switch to a gentler reagent. Ensure complexes are made in serum-free medium.

Biological Roles

Item-specific target

• Target indicated by product name: human CERS6 (ceramide synthase 6). No additional identifiers are specified for this item; consult CoA/Spec Sheet for sequence/ID details.

General/literature biology of CERS6

• Function: CERS6 is an ER-resident acyl-CoA–dependent ceramide synthase that preferentially generates C16 ceramide species from sphinganine/sphingosine backbones (literature).
• Pathway: Central to de novo sphingolipid biosynthesis, influencing levels of ceramides, sphingomyelins, and complex glycosphingolipids.
• Cellular processes: Ceramides modulate membrane microdomains, vesicular trafficking, apoptosis, stress responses, and mitochondrial dynamics. CERS6 activity impacts mitochondrial fission through ceramide-dependent signaling (literature).
• Expression: CERS isoforms show tissue-specific expression; CERS6 is detectable in multiple human tissues and many cultured cell lines (literature).
• Knockdown outcomes: Reduced CERS6 commonly decreases C16-ceramide pools and can alter cell morphology, migration, and sensitivity to stress; precise phenotypes are cell-type dependent and should be experimentally verified (literature; avoid clinical claims).

Use of siRNA against CERS6

• Enables time-resolved interrogation (24–96 h) of sphingolipid flux by LC–MS profiling.
• Facilitates genetic–chemical interaction studies with ceramide metabolism inhibitors to map pathway redundancies.

Buffer Applications

Applicability

• Relevant primarily for reconstitution, storage, and delivery of siRNA to cells.

General/literature guidance

• Reconstitution buffer: RNase-free water (preferred for versatility) or 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) to improve long-term stability by chelating divalent cations.
• Working buffer/media: Complex siRNA with transfection reagents in serum-free, low-ionic-strength media (e.g., Opti-MEM) according to the reagent’s protocol; add complexes to cells in complete medium after 10–30 min complexation.
• pH range: Maintain near-neutral pH (7.0–8.0). Avoid repeated freeze–thaw of buffered stocks.
• Sterility: If sterility is required, sterile-filter with 0.22 µm low-binding filters. Do not autoclave oligonucleotide solutions.
• Divalent cations: Minimize Mg2+/Ca2+ during complex formation to avoid aggregation; normal levels in culture medium post-delivery are acceptable.
• Storage aliquots: Prepare single-use aliquots to avoid RNase introduction during repeated handling.

Green Alternatives

Context

• This product is an aqueous oligonucleotide reagent used at micromolar concentrations; its use phase generally avoids volatile organic solvents. Environmental burden is primarily from single-use plastics and transfection reagents.

Best practices (literature/practice)

• Solvent footprint: Reconstitute and dilute in water or buffered saline; avoid alcohols or chlorinated solvents. Prepare concentrated stocks to reduce plastic usage.
• Transfection reagents: Prefer lower-toxicity, biodegradable lipid/polymer formulations when available; minimize reagent-to-siRNA ratios while maintaining efficacy.
• Energy use: Store at -20°C as specified; consolidate freezer space and use insulated secondary containers to minimize frost formation and energy waste.
• Waste reduction: Utilize multi-well formats efficiently; pool replicate transfections to reduce consumables. Dispose of nucleic acid waste via aqueous streams where permitted.

Comparison (general)

• siRNA vs plasmid/shRNA: Transient siRNA avoids antibiotic selection and bacterial culture waste; however, repeated dosing may be needed. shRNA can reduce reagent use long-term but introduces viral vector considerations.
• Chemical synthesis: If choosing modified siRNA (2′-OMe/PS), note that synthesis has a larger upstream chemical footprint; balance with increased potency/longevity that can reduce experimental repeats.

Pharmaceutical Uses

Item-specific status

• This product is labeled for research use only. It is not intended for diagnostic, therapeutic, or GMP manufacturing purposes.

General context (literature)

• Oligonucleotides similar to siRNA can serve as drug substance/excipients in regulated settings only when manufactured under GMP with stringent controls on identity, purity, impurities (e.g., shortmers), endotoxin, and bioburden.
• Formulation learnings (non-clinical lab context): Complexation with cationic lipids/polymers protects siRNA from nuclease degradation and facilitates endosomal escape; parameters such as N/P ratio, particle size (~80–150 nm), and zeta potential are tuned for delivery. These considerations can inform research formulations but do not imply suitability for human or veterinary use.
• Pharmacopeial status: There is no monograph universally covering sequence-specific siRNA; specifications are sequence- and platform-dependent. Always segregate research-use materials from any translational or clinical development streams.

Physical Properties

Item-specific properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight (per duplex), extinction coefficient, and exact composition: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values for siRNA (sequence-dependent; provided as context only)

• Typical length: 21±2 nucleotides per strand with 2-nt 3′ overhangs.
• Approximate MW: ~13–15 kDa per duplex for 21-mers; varies with sequence and modifications (literature).
• Solubility: Freely soluble in nuclease-free water and low-ionic-strength buffers (e.g., 10 mM Tris, pH 7.4–8.0) (literature).
• pKa: Nucleobase ionizations are complex; duplex overall behaves as a polyanion (phosphate pKa1 ~1–2, pKa2 ~6–7 for monomeric phosphates; duplex remains negatively charged at physiological pH) (literature).
• LogP: Not meaningful for polyanionic oligonucleotides; they are highly hydrophilic (literature).
• Thermal properties: Strand Tm depends on sequence, length, salt, and modifications; 21-mer duplexes often melt in the 60–85°C range in 1 M Na+; significantly lower in low salt (literature).
• Storage form: Often supplied dried or in aqueous RNase-free solution; hygroscopicity minimal compared with small molecules but aqueous solutions are prone to hydrolysis/RNase degradation (literature).

Quality and Grades

Item-specific details

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• Stabilizers/modifications: Not specified for this item; refer to CoA/Spec Sheet.

General notes on siRNA quality (literature/practice)

• Purity levels: Common offerings include desalted, cartridge-purified, HPLC-purified, or PAGE-purified. Higher purity reduces truncated species and can improve potency and reproducibility, especially at low nanomolar doses.
• Identity confirmation: Typically by MALDI-TOF/ESI-MS and, for high-spec products, by analytical HPLC or capillary electrophoresis.
• Functional QC: Potency assessed by target knockdown in a standard cell line; absence of immunostimulatory motifs and minimized off-target seed effects are common design goals.
• RNase-free assurance: Materials, buffers, and containers should be certified RNase/DNase-free. Endotoxin testing may be provided for sensitive applications.
• Set A concept: Pre-designed sets usually contain multiple distinct siRNA duplexes per gene to allow selection of the most effective sequence and to enable pooled or sequential knockdown strategies to mitigate off-target effects.
• Documentation: For sequence, purity, mass, and analytical traces, consult the item’s CoA/Spec Sheet.

Reaction and Applications

Item-specific scope

• Intended use: RNA interference (RNAi)-mediated knockdown of human CERS6 transcripts in cultured cells (inferred from product name). Specific sequences and recommended use conditions are not specified for this item; refer to CoA/Spec Sheet.

General/literature applications for pre-designed siRNA

• Target validation: Transiently reduce CERS6 expression to probe roles in sphingolipid biosynthesis and downstream phenotypes.
• Pathway dissection: Evaluate impacts on ceramide species composition, mitochondrial dynamics, autophagy markers, or stress signaling.
• Phenotypic screens: Use pooled siRNA (from the set) for robust knockdown, then deconvolute to single duplexes to confirm on-target effects.
• Rescue experiments: Combine siRNA with siRNA-resistant cDNA to confirm specificity.
• Orthogonal modalities: Cross-validate with CRISPRi/ASOs to strengthen conclusions.

Practical tips (literature)

• Use multiple siRNA sequences per target to control for off-targets and sequence-specific toxicity.
• Optimize dose (e.g., 5–50 nM final) and transfection reagent according to cell line; titrate to minimize cytotoxicity while achieving ≥70% mRNA knockdown at 24–72 h.
• Include controls: non-targeting siRNA, positive control siRNA (e.g., to a housekeeping gene), and mock transfection.
• Validate: qRT-PCR for mRNA, Western blot for protein levels, and functional readouts relevant to ceramide metabolism.
• RNA quality: Maintain RNase-free conditions; prepare fresh complexes each use.

Reaction Conditions

Applicability

• Here, “reaction” refers to cellular delivery and gene silencing rather than a chemical transformation. The following are general literature guidelines; optimize per cell line and transfection reagent. No item-specific protocol is provided—refer to CoA/Spec Sheet if available.

Typical transfection conditions (literature)

• Cell density: Plate cells to reach 40–70% confluence at transfection.
• siRNA dose: 5–50 nM final; start at 10 nM and titrate. For pooled sets, maintain constant total siRNA by equimolar mixing.
• Complex formation: Dilute siRNA in serum-free medium (e.g., Opti-MEM). Separately dilute transfection reagent. Combine at recommended ratios (e.g., 1–3 µL lipid per 10 nM siRNA in 24-well; consult reagent datasheet). Incubate 10–20 min at RT.
• Incubation: Add complexes dropwise to cells. Replace with fresh complete medium after 4–6 h if cytotoxicity is observed; many systems allow continuous exposure.
• Readouts: qRT-PCR at 24–48 h for mRNA knockdown; Western blot at 48–96 h for protein reduction. Functional assays as appropriate.
• Controls: Non-targeting siRNA, positive knockdown control, and transfection-only control. Include an siRNA-resistant rescue construct when feasible.
• Expected outcomes: Well-optimized systems often achieve >70% mRNA knockdown with minimal viability loss (literature; actual performance depends on sequence and cell type).

Safety and Handling

Item-specific safety information

• GHS classification, signal word, H-statements, pictograms: Not specified for this item; refer to SDS.
• Research use: For research use only (provided).
• Storage conditions (provided): Store at -20°C.

General/literature guidance for siRNA oligonucleotides

• Hazard profile: Synthetic nucleic acids are generally considered low hazard; treat as laboratory chemicals and avoid inhalation/ingestion or contact with eyes/skin. Always follow institutional biosafety practices and consult the SDS for authoritative data.
• PPE: Lab coat, safety glasses, and disposable gloves. Change gloves frequently to maintain RNase-free technique.
• Handling precautions: Work in a clean area with RNase-free tools and plastics. Use dedicated RNase-free tips/tubes. Avoid generating aerosols.
• Incompatibilities: Avoid strong acids/bases at elevated temperature (accelerates depurination/backbone cleavage). Avoid ubiquitous RNases; decontaminate benches and equipment with RNase-inactivating agents.
• First-aid (overview; defer to SDS): If on skin/eyes, rinse with water. If inhaled, move to fresh air. If ingested, rinse mouth with water. Seek medical advice if symptoms occur.
• Waste: Dispose of aqueous oligonucleotide solutions in accordance with local regulations; decontaminate RNase-containing waste appropriately.

Solvent Selection

Applicability

• As an oligonucleotide reagent, this product is reconstituted in aqueous, RNase-free media rather than organic solvents.

General guidance (literature/practice)

• Primary solvents: RNase-free water, or 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) to chelate divalent cations that catalyze hydrolysis. For in vitro cell work, reconstitute in water and dilute into serum-free medium for complexation with transfection reagent.
• Ionic strength and pH: Maintain near-neutral pH (7.0–8.0). High salt increases duplex stability; however, complexation protocols with lipids/polymers often specify low-salt media (Opti-MEM or similar) during complex formation.
• Avoid: Organic solvents (alcohols, DMSO) are generally unnecessary and can impair transfection complex formation and cell viability. If sterility is required, sterile-filter using low-protein-binding 0.22 µm filters; avoid heat sterilization.
• Concentration considerations: Prepare a concentrated stock (e.g., 10–100 µM) in RNase-free water for minimal ionic carryover; dilute immediately before use into transfection mix.
• Adsorption losses: Minimize by using low-binding tubes and keeping concentrations ≥1–2 µM. Brief heating (≤55°C for 5 min) can aid dissolution without degrading the oligo.

Storage and Reconstitution

Item-specific conditions

• Storage: Store at -20°C (provided).
• Shipping: Shipped in ice chest + ice pads (provided).
• Appearance/formulation on receipt: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance for siRNA

• Upon receipt: If supplied dry, briefly centrifuge to collect material. If supplied in solution, verify volume and concentration.
• Reconstitution: Use RNase-free water for a convenient 10–100 µM stock. For long-term stability, 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0) can be used; note that EDTA may be incompatible with some downstream enzymatic assays.
• Aliquoting: Prepare single-use aliquots (e.g., 5–20 µL) to avoid freeze–thaw cycles.
• Storage temps: Store working stocks at -20°C (days–weeks). For extended storage, -80°C is advisable (literature). Avoid repeated freeze–thaw; thaw on ice and keep cold during handling.
• Light and nuclease protection: Protect from prolonged ambient light; always use RNase-free plastics and change gloves frequently.
• Stability expectations: Properly stored, dried siRNA is stable for many months at -20°C; aqueous stocks remain stable for several months at -20°C and longer at -80°C (literature). Always confirm integrity by functional assay or analytical methods as needed.

Structure and Identity

Item-specific facts (from Product Data)

• Product: CERS6 Human Pre-designed siRNA Set A (SKU: C1466355)
• Intended target: human CERS6 mRNA (per product name)
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Sequence(s), length, strand count, and chemical modifications: Not specified for this item; refer to CoA/Spec Sheet.
• CAS, CID, InChIKey, SMILES: Not specified for this item; refer to CoA/Spec Sheet.

General/literature description of structure (for siRNA)

• siRNA are typically double-stranded RNA duplexes (~19–27 bp) with 2-nt 3′ overhangs and 5′-phosphate/3′-hydroxyl termini (literature).
• Functional features: antisense (guide) strand complementary to target mRNA; sense (passenger) strand. May include chemical stabilizations (e.g., 2′-OMe, 2′-F, phosphorothioates) to enhance stability and reduce off-targets (literature).
• Structural motif: A-form RNA helix; sequence-specific base pairing via Watson–Crick interactions (literature).

Synthetic Utility

Scope

• As a functional genomics reagent, siRNA has no conventional role as a building block in chemical synthesis. Instead, it is a tool for perturbing gene expression to support synthetic biology and pathway engineering studies.

Utility in research workflows (literature)

• Retrosynthetic planning aid: Gene knockdown can reveal whether a biosynthetic or metabolic transformation is rate-limiting, guiding which enzymes/pathways to target for engineering or inhibitor design.
• Probe in chem-bio screens: Combine siRNA with small-molecule libraries to discover synthetic lethal or pathway-sensitizing interactions.
• Tool for target deconvolution: Knock down CERS6 to confirm whether phenotypes induced by putative ceramide-pathway modulators are on-target.
• Systems-level integration: Pair with lipidomics and transcriptomics to map network responses to altered ceramide synthesis.

Practical considerations

• Use multiple independent siRNA sequences (as in a "Set A") to ensure that observed effects are robust to sequence choice.
• Time windows: Most protein knockdown phenotypes manifest 48–96 h post-transfection, depending on protein stability and turnover. Adjust sampling accordingly.

Target Specificity

Item-specific information

• Target designation per product name: Human CERS6 (ceramide synthase 6). Set A implies multiple pre-designed siRNA duplexes targeting distinct regions of the CERS6 transcript. Additional details (sequences, target exons/isoforms, off-target analysis) are not specified for this item; refer to CoA/Spec Sheet.

General/literature notes on specificity

• Mechanism: The antisense strand loads into RISC and guides sequence-specific cleavage of complementary CERS6 mRNA. Seed-region complementarity (positions 2–8) can mediate off-target repression; using multiple independent siRNA reduces the likelihood of shared off-targets.
• Isoforms: If CERS6 has multiple transcript variants, design typically targets exons common to major isoforms; verify isoform expression in your cells.
• SNPs and species: Human-specific siRNA may not efficiently target orthologous rodent transcripts due to sequence divergence; align to the intended species transcriptome to ensure compatibility.
• Verification: Confirm on-target knockdown with at least two duplexes and rescue by siRNA-resistant cDNA. Assess off-targets via RNA-seq or seed enrichment analyses when critical.