Ep300 Rat Pre-designed siRNA Set A

Cat. No.: E1479702

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

Ep300 Rat Pre-designed siRNA Set A contains three designed siRNAs for Ep300 gene (Rat), as well as a negative control, a positive control, and a FAM-labeled negative control. Components Ep300 siRNA-1: 5 nmol (HPLC) Ep300 siRNA-2: 5 nmol (HPLC) Ep300 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

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Product Line

Biological Products

Application Protocols

No item-specific, validated application protocols are provided for this product. The following is general guidance for siRNA-mediated knockdown; optimize per cell type and transfection reagent.

General lipid-mediated transfection (literature):

1. Plate cells to reach ~40–50% confluence at transfection.
2. Dilute siRNA in reduced-serum medium to achieve a desired final concentration (start 10–20 nM).
3. In a separate tube, dilute transfection reagent per vendor instructions.
4. Combine diluted siRNA and reagent; incubate 10–20 min at room temperature to form complexes.
5. Add complexes dropwise to cells; gently rock plate. Optional: replace with complete medium after 3–6 h.
6. Harvest RNA at 24–48 h; harvest protein/functional readouts at 48–96 h.

Electroporation (general):

• Mix cells with siRNA in device-specific buffer; use recommended program for the cell type. Immediately transfer to pre-warmed medium.

Controls and analytics:

• Include non-targeting siRNA, positive control siRNA, and cytotoxicity assessment (e.g., CellTiter). Validate knockdown by qRT-PCR and Western blot for Ep300.

Note: For exact duplex sequences, recommended working concentrations, and reagent compatibility specific to “Set A”, consult the CoA/Spec Sheet.

Biological Roles

Target overview (general biology of Ep300 in rat):

• Ep300 encodes the transcriptional coactivator p300 (KAT3B), a lysine acetyltransferase that acetylates histones (e.g., H3K27, H3K18) and numerous non-histone substrates, facilitating chromatin relaxation and transcriptional activation.
• p300 functions as a scaffold integrating sequence-specific transcription factors (e.g., p53, HIF1A, CREB/CBP partners) with the basal transcriptional machinery and chromatin remodelers. It cooperates closely with CREBBP (CBP), with partial redundancy and distinct target preferences.
• Pathways: DNA damage response (p53 acetylation), hypoxia signaling (HIF1A coactivation), hormonal and developmental programs, immune and inflammatory responses via NF-κB and AP-1 family members.
• Cellular roles: Enhancer activation, super-enhancer maintenance, cell cycle regulation, differentiation, and metabolic adaptation through acetylation-dependent modulation of TFs and co-regulators.

Relevance of knockdown (literature rationale):

• Reducing Ep300 expression typically decreases global and locus-specific histone acetylation marks associated with active enhancers, dampening transcription from responsive promoters/enhancers.
• Phenotypic outcomes are cell-type and context dependent; compensation by CBP (Crebbp) may occur. Dual targeting strategies are sometimes employed to probe redundancy.

Note: The above describes general biology of the target gene/protein and is not a medical/clinical claim. This product is designated for research use only.

Buffer Applications

Buffer formulation is relevant for siRNA stability and transfection performance rather than classical acid–base buffering of reactions.

General guidance (literature):

• Stock reconstitution: RNase-free water is commonly used for immediate use. For longer-term storage, 1× or 0.1× TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.0) helps chelate divalent cations and suppress nuclease activity.
• Working dilutions: Prepare in nuclease-free water or the diluent recommended by your transfection reagent (often reduced-serum medium like Opti-MEM). Avoid high-salt buffers during complex formation with cationic lipids/polymers.
• Handling: Keep solutions on ice during setup. Use low-binding, RNase-free tubes and tips. Filter sterilization is generally unnecessary if using sterile components and good asepsis.
• Electroporation: For primary cells or suspension lines, use manufacturer-provided electroporation buffers; compatibility with siRNA is typically high, but ionic strength/osmolality must match device requirements.

Item-specific buffer specifications (pH, salt, stabilizers) are not provided for this product. For any mandatory buffer composition or concentration, refer to the CoA/Spec Sheet.

Green Alternatives

This siRNA reagent is aqueous and used at low mass/volume, so traditional solvent “greenness” comparisons are not central. Nevertheless, greener practice focuses on minimizing hazardous reagents and plastic waste.

Greener practice suggestions (general):

• Use RNase-free water or TE instead of organic co-solvents. Avoid phenol/chloroform for RNA handling wherever possible.
• Select transfection methods compatible with serum and standard media to reduce wash steps and solvent consumption.
• Scale appropriately to limit single-use plastic; employ multi-channel pipetting and reusable RNase-free racks where permitted by contamination control policies.
• Opt for low-toxicity, biodegradable transfection reagents when possible; evaluate electroporation settings to reduce chemical use for hard-to-transfect cells.

Alternative platform comparisons (general, not item-specific):

• siRNA vs. shRNA: siRNA offers transient, rapid knockdown with minimal vector waste; shRNA requires plasmids/viral packaging (higher waste/biological containment burdens) but enables stable suppression.
• siRNA vs. CRISPRi: CRISPRi (dCas9-KRAB) affords stable transcriptional repression with a single guide and no RNA duplex consumption; however, it requires cell engineering and may have off-target chromatin effects. siRNA remains simpler and chemical-only, with reduced long-term biosafety concerns.

Pharmaceutical Uses

This product is for research use only and is not intended for human or veterinary use.

General formulation/manufacturing context for siRNA tools (literature):

• siRNA duplexes are widely used as development tools in discovery labs to validate targets and to benchmark pharmacological modulation in cell systems before medicinal chemistry or biologic campaigns. They inform manufacturability and process-relevant assays (e.g., reporter potency, pathway knockdown) but are not GMP materials.
• Excipients for laboratory siRNA handling commonly include RNase-free water or TE for stock solutions. For in vitro delivery, cationic lipids/polymers or electroporation buffers are used; these are process aids rather than formulation excipients.
• Regulatory status: Research-grade siRNA is not covered by pharmacopeial monographs and is unsuitable for clinical or diagnostic applications without further qualification and manufacturing controls.
• For groups transitioning toward therapeutic modalities, orthogonal approaches (CRISPRi, ASOs) are often evaluated in parallel at research scale; however, this product should not be used for any clinical development activities.

Physical Properties

Item-specific physical properties are not provided for this siRNA set.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: siRNA is water-soluble in nuclease-free aqueous buffers (general, literature). Typical reconstitution uses RNase-free water or low-ionic-strength TE pH ~7.5–8.0.
• pKa, logP, refractive index, density, BP/MP: Not typically defined for oligonucleotide mixtures; Not specified for this item; refer to CoA/Spec Sheet.

General/literature notes for siRNA:

• siRNA duplexes dissolve readily in RNase-free water to form clear solutions at µM–mM stock concentrations, depending on length and counterion form (e.g., sodium salt).
• UV absorbance at 260 nm follows Beer–Lambert law; extinction coefficients are sequence-dependent and normally provided per sequence on the CoA. If required, calculate with standard nearest-neighbor models (literature) and verify against supplier data.
• Lyophilized siRNA typically equilibrates quickly in aqueous solvents; gentle mixing (no vortexing of highly concentrated stocks) and short low-speed spin are recommended to collect contents and facilitate dissolution (general guidance).

Quality & Grades

Item-specific grade/purity is not provided for this siRNA set.

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

General considerations for siRNA quality (literature/practice):

• Purification levels commonly include desalted, cartridge-purified, or HPLC/ PAGE-purified duplexes. HPLC or PAGE purification can reduce truncated species and improve knockdown consistency, particularly for longer or modified oligos.
• Identity/Integrity: Suppliers typically confirm by MALDI-TOF/ESI-MS and assess duplex annealing by native PAGE or equivalent. OD260 and calculated nmol/µg values are used to quantify.
• Endotoxin and bioburden: For cell-based assays, low endotoxin is preferred; specifications, if any, are product-specific (refer to CoA/SDS).
• Stabilizers/Modifications: Some siRNAs include chemical modifications (e.g., 2′-OMe, 2′-F, phosphorothioates) to enhance nuclease resistance and reduce off-target effects; presence/absence is item-specific (not provided here).
• What to check on receipt: Verify target gene (Ep300, rat), sequences (if provided), amount (nmol/OD units), recommended resuspension solvent, and any stated QC metrics on the accompanying documentation.

Reaction & Applications

Applications here refer to functional genomics experiments rather than chemical reactions.

• Primary application: Sequence-specific knockdown of rat Ep300 transcripts via RNA interference (RNAi). The antisense (guide) strand is incorporated into RISC, directing endonucleolytic cleavage or translational repression of Ep300 mRNA (literature mechanism).
• Experimental contexts: Phenotypic screening, pathway dissection (e.g., p53, HIF1A, CREB-dependent transcription), validation of Ep300-dependent reporter assays, and combination studies with chemical inhibitors or CRISPR knockout for orthogonal confirmation.
• Readouts: qRT-PCR for mRNA reduction, Western blot/ELISA for EP300 protein, ChIP-qPCR for histone acetylation marks (e.g., H3K27ac) at target promoters/enhancers, functional assays for transcriptional activity.
• Controls: Non-targeting siRNA, positive control siRNA (to a housekeeping gene), and rescue experiments with siRNA-resistant Ep300 cDNA (silent mutations) to verify on-target effects.
• Practical tips: Perform small-scale transfection optimization (cell density, reagent type/ratio, serum presence, exposure time) before full experiments. Include at least two independent siRNA duplexes to confirm phenotypes (general best practice). Harvest at multiple time points (24–96 h) to capture transcript and protein kinetics.
• Off-target mitigation: Use minimal effective dose (typically 5–20 nM final, literature), chemical modifications (if available), and pooled duplex strategies; validate by transcriptome profiling when critical.

Reaction Conditions

Here “reaction conditions” refers to transfection and knockdown conditions in cell-based assays (general literature guidance; optimize per cell type).

• Cell density at transfection: Typically 30–60% confluence for adherent cells to balance viability and uptake; suspension cells at mid-log phase.
• siRNA working concentration: Commonly 5–50 nM final; start with 10–20 nM and titrate downward to minimize off-target effects while retaining potency.
• Complexation: Dilute siRNA and lipid/polymer separately in serum-free or reduced-serum medium (e.g., Opti-MEM), combine at vendor-recommended ratios, incubate 10–20 min at room temperature to form complexes.
• Incubation: Add complexes to cells, optionally replace with complete medium after 3–6 h for sensitive cells. Many lipid reagents permit serum presence throughout—verify with reagent guidance.
• Readout timing: mRNA nadir often at 24–48 h; protein nadir at 48–96 h depending on target half-life. For p300-related chromatin marks, allow 48–72 h before ChIP.
• Replicates/controls: Include non-targeting and positive control siRNAs; perform at least biological triplicates.
• Electroporation: For primary/“difficult” cells, use manufacturer-specific programs and buffers; typical siRNA amounts range 50–300 pmol per cuvette depending on volume and device.

Note: No item-specific transfection reagent or conditions are provided; refer to CoA/Spec Sheet for any product-specific recommendations if available.

Safety & Handling

Item-specific hazard classification is not provided.

• GHS classification, signal word, hazard (H) statements, pictograms: Not specified for this item; refer to SDS.

General safety for RNA oligonucleotides (literature/practice):

• siRNA duplexes are generally considered low hazard in typical laboratory handling. Avoid inhalation of powders/aerosols and contact with eyes or skin. Use standard laboratory PPE: lab coat, gloves (nitrile), and safety glasses.
• Critical incompatibilities: RNases. Maintain RNase-free technique to protect product integrity (use RNase-free consumables; decontaminate surfaces with RNase inhibitors; avoid touching tips/tubes with bare hands).
• First-aid overview: If skin or eye contact occurs, rinse with water for several minutes; remove contaminated clothing. If inhaled (powder), move to fresh air. If ingested, rinse mouth with water. Seek medical advice if symptoms persist. Always defer to the product SDS.
• Handling tips: Work on ice for prolonged manipulations to limit nuclease activity; minimize freeze–thaw cycling by aliquoting. Use dedicated RNase-free pipettes/tips. Avoid microbial contamination.
• Waste: Dispose of solutions and consumables as non-hazardous laboratory biological/chemical waste per institutional policy unless mixed with hazardous reagents (follow local regulations).

Solvent Selection

This product is an siRNA reagent; organic solvent selection is not typically applicable. Use nuclease-free aqueous media.

General guidance (literature):

• Primary solvents: RNase-free water, 1× or 0.1× TE buffer (10 mM Tris, 0.1–1 mM EDTA, pH 7.5–8.0). TE provides chelation of divalent cations to suppress RNase activity.
• Working buffers/media: For complexation with transfection reagents, reduced-serum medium (e.g., Opti-MEM) is commonly used during complex formation; standard complete culture medium is restored after 3–6 h (protocol-dependent).
• Avoid: Phenol, chloroform, and alcohols can denature or precipitate RNA; DEPC-treated water must be fully autoclaved to decompose residual DEPC before use. High-salt buffers can affect complexation with cationic lipids/polymers.

Comparison (general):

• RNase-free water: Maximizes simplicity and compatibility; best for short-term stocks used promptly.
• 1× TE pH ~8.0: Enhances stability for long-term frozen storage; EDTA chelates Mg2+/Ca2+, reducing nuclease activity.
• PBS: Acceptable for some applications but may decrease complexation efficiency with certain transfection reagents; evaluate empirically.

Storage & Reconstitution

Item-specific storage is provided; detailed reconstitution parameters are not.

• Storage conditions (from Product Data): Store at -20°C. Avoid repeated freeze–thaw cycles.
• Shipping (from Product Data): Shipped in ice chest with ice pads to maintain cold chain.
• Research use note: For research use only.

Not specified for this item; refer to CoA/Spec Sheet:

• Recommended reconstitution solvent, target stock concentration, and any stabilizers/additives.

General guidance (literature/practice):

• Upon receipt, briefly centrifuge tubes to collect lyophilized material. Reconstitute in RNase-free water or 1×/0.1× TE (pH 7.5–8.0). Typical stock concentrations are in the 10–100 µM range; choose based on dosing needs and volume constraints.
• Mix gently by pipetting; avoid vigorous vortexing of highly concentrated stocks. Allow to sit on ice for 10 minutes to ensure complete dissolution.
• Aliquot into RNase-free, low-binding tubes to single-use portions; store at -20°C for medium term or -80°C for long term. Protect from repeated freeze–thaw by using small aliquots.
• Thaw on ice; keep solutions cold during setup. Discard aliquots showing turbidity or contamination. Maintain RNase-free technique at all times.

For any product-specific constraints (e.g., presence of chemical modifications, desalting requirements, or annealed vs. single-stranded format), consult the CoA/Spec Sheet.

Structure & Identity

Item-specific identifiers provided: SKU E1479702; Product Name Ep300 Rat Pre-designed siRNA Set A. No chemical identifiers are provided for this item.

• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• CAS, CID, SMILES, InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
• Target: Rat Ep300 (E1A binding protein p300; KAT3B homolog in Rattus norvegicus).

General structural description (literature/general knowledge):

• siRNA reagents are typically short, double-stranded RNA duplexes (commonly 19–21 bp) with 2-nt 3′ overhangs and 5′-phosphate/3′-hydroxyl termini; strands are composed of ribonucleotides and may contain optional chemical stabilizations to reduce nuclease susceptibility.
• The sense strand is complementary to the mRNA target’s coding sequence; the antisense (guide) strand is loaded into RISC to direct sequence-specific cleavage or translational repression of the target transcript.
• Pre-designed “Set A” nomenclature usually denotes a collection of distinct, non-overlapping duplexes to the same gene to facilitate screening and confirmation of on-target phenotypes (number, sequences, and modifications are product-specific and not provided here).

Synthetic Utility

Not a chemical building block. Synthetic organic transformations are not applicable.

Research utility (general for siRNA):

• Tool compound for functional genomics: transient gene silencing to dissect Ep300-dependent pathways, validate phenotypes, and generate hypothesis-driven SAR for small-molecule screening against p300/CBP functions.
• Mechanistic interrogation: Differentiate catalytic vs. scaffolding roles of p300 by combining siRNA-mediated depletion with catalytic inhibitors and rescue constructs.
• Epistasis mapping: Pair Ep300 knockdown with perturbations to TFs (e.g., HIF1A, TP53, CREB1) to position p300 within regulatory hierarchies.
• Chromatin studies: Assess enhancer competence and histone acetylation dynamics upon reduced p300 levels using ChIP-seq/ATAC-seq; integrate with RNA-seq for transcriptional outcomes.

Note: For chemical synthesis applications, this section is not applicable; focus on cell/molecular biology use cases above.

Target Specificity

Item-specific targeting details beyond the product name are not provided.

• Declared target: Rat Ep300 (E1A binding protein p300; also known as KAT3B). Species: Rattus norvegicus.
• Duplex design: Pre-designed siRNA sets typically comprise multiple non-overlapping duplexes to reduce the risk of single-sequence idiosyncrasies and to confirm on-target phenotypes (general). Exact sequences, lengths, chemical modifications, and the number of duplexes in “Set A” are not specified for this item; refer to CoA/Spec Sheet.
• Off-target considerations (general): Algorithms usually avoid seed matches in 3′UTRs of unrelated transcripts, filter SNP regions, and control GC content for efficacy vs. toxicity balance. Seed-region off-targets can be mitigated by dose reduction and chemical modifications (if present).
• Validation strategy: Demonstrate target suppression by qRT-PCR and Western blot; confirm phenotype with at least two independent duplexes and, ideally, a rescue construct bearing silent mutations in the siRNA target site.

Frequently Asked Questions

How should this product be stored?

Store at ?20 °C. Freezer storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.