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Application Protocols
The following protocol outlines general, literature‑based steps for siRNA transfection and knockdown assessment for Ddr1 in mouse cells. It is provided as guidance and is not item‑specific.
A. Preparation
Thaw siRNA on ice. Briefly spin to collect contents. Work RNase‑free.
Prepare stock at 20–50 µM in RNase‑free water or TE‑Low. Aliquot to avoid freeze–thaw.
B. Transfection (adherent cells, 24‑well example)
Seed 5×10^4–8×10^4 cells/well 18–24 h prior to reach ~50% confluence at transfection.
For 10 nM final siRNA: Dilute 0.5–1 µL of 20 µM siRNA in 50 µL Opti‑MEM; in a separate tube, dilute lipid reagent per manufacturer (e.g., 1–2 µL in 50 µL). Incubate 5 min.
Combine, mix gently, incubate 10–20 min at RT to form complexes.
Add complexes dropwise to wells containing 400 µL complete medium. Gently rock plate.
Optional: Replace medium after 4–6 h if toxicity observed; otherwise maintain.
C. Knockdown assessment
24–48 h: Isolate RNA and quantify Ddr1 mRNA by RT‑qPCR (normalize to housekeeping genes).
48–96 h: Harvest protein; immunoblot for DDR1 total and phospho‑DDR1 after collagen stimulation. Assess downstream markers (e.g., p‑ERK) as functional readouts.
Controls and troubleshooting
Include non‑targeting siRNA and mock transfection.
If knockdown is suboptimal, titrate siRNA (5–25 nM), adjust lipid ratio, verify cell health, and confirm sequence identity.
Item‑specific validated protocols or recommended transfection reagents: Not specified for this item; refer to CoA/Spec Sheet.
Biological Roles
Target overview (literature, general information)
Ddr1 (Discoidin domain receptor 1) is a collagen‑activated receptor tyrosine kinase expressed in various epithelial and mesenchymal tissues in mouse and other vertebrates. Upon binding fibrillar and non‑fibrillar collagens, DDR1 undergoes autophosphorylation and initiates signaling cascades influencing cell adhesion, ECM remodeling, migration, and differentiation.
Downstream signaling: DDR1 activation engages pathways such as MAPK/ERK, PI3K/AKT, and STAT, and modulates matrix metalloproteinases and integrin dynamics (context‑dependent).
Developmental and homeostatic roles: DDR1 contributes to tissue morphogenesis and epithelial polarity and participates in kidney and lung biology in animal models.
Reagent role (literature, general)
siRNA‑mediated knockdown of Ddr1 enables transient suppression of DDR1 signaling to dissect its contribution to ECM‑sensing pathways, crosstalk with integrins, and regulation of actin cytoskeleton organization. Combining siRNA with phosphoproteomics or transcriptomics can map DDR1‑dependent nodes.
Experimental considerations
Because DDR1 is activated by collagen, consider plating substrates (e.g., collagen I/IV coatings) and time‑resolved stimulation when evaluating functional impacts of Ddr1 knockdown.
Validate knockdown specificity with multiple duplexes, rescue experiments using siRNA‑resistant Ddr1 cDNA, and pathway readouts (e.g., phospho‑DDR1, phospho‑ERK).
Item-specific biological validation or expression datasets: Not specified for this item; consult CoA/Spec Sheet.
Buffer Applications
This product is an siRNA reagent rather than a classical buffer component; it is generally used within RNase‑free aqueous buffers for reconstitution, storage, and transfection complexation.
Practical buffer guidance (general, literature):
Reconstitution: RNase‑free water or TE‑Low (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0). EDTA chelates divalent cations that can support RNase activity; avoid EDTA in final transfection mixes if it interferes with complex formation.
Working dilutions: Prepare in Opti‑MEM or similar reduced‑serum medium for complexation with cationic lipids or polymers per reagent instructions.
Storage buffer: TE‑Low is commonly used for stock stability at −20 °C; aliquot to avoid freeze–thaw.
Nuclease control: Clean surfaces with RNase decontamination solutions; use certified RNase‑free tips/tubes.
Not typically applicable: Classical buffering capacity calculations, pKa windows, and electrophoresis buffer recipes are not central to this product’s use. For electrophoretic analysis (optional), native or denaturing RNA gels can be prepared with TBE/TBE‑Urea (general), but this is ancillary to transfection workflows.
Item-specific buffer composition or recommended formulations: Not specified for this item; refer to CoA/Spec Sheet.
Green Alternatives
For siRNA reagents, environmental impact centers on buffer systems, plastics usage, and transfection chemistries rather than choice of organic solvent.
Greener practice suggestions (general):
Use aqueous, nuclease‑free buffers (water or TE‑Low) and avoid unnecessary organic co‑solvents.
Select transfection reagents with lower cytotoxicity and reduced required dose to minimize waste; consider biodegradable or lipid‑based systems with favorable safety profiles.
Scale reactions appropriately to reduce consumables and reagent waste; pool replicate analyses to minimize multiple freeze–thaw cycles and aliquot waste.
Implement cold‑chain efficiency: consolidate shipments and choose reusable ice packs/dry ice alternatives when validated by the supplier.
Decontaminate and segregate nucleic‑acid waste for appropriate disposal to prevent environmental RNase contamination and ensure compliance with local regulations.
Comparison of common media choices (general)
Water vs TE‑Low: TE‑Low may extend shelf life, reducing waste from degraded stocks, at the cost of introducing minimal buffer components.
Serum‑free complexation vs in‑medium complexation: Forming complexes in small volumes of serum‑free medium can reduce reagent consumption while maintaining efficacy.
Item-specific sustainability certifications or green attributes: Not specified for this item.
Pharmaceutical Uses
This product is designated for research use only and is not intended for diagnostic, therapeutic, or clinical applications.
General formulation context for siRNAs (literature; not specific to this item):
Excipient roles: In pharmaceutical development, siRNAs often require specialized delivery systems (lipid nanoparticles, conjugates) and stringent quality attributes (purity, identity, low endotoxin). Such considerations are beyond the scope of standard research‑grade reagents.
Stability: Lyophilization with stabilizers (e.g., sugars, buffers) can improve shelf life; however, no excipients or formulation details are specified for this item.
For this catalog item
No pharmacopeial status, GMP grade, clinical verification, or therapeutic claims are made or implied.
Any use in humans or animals for therapeutic purposes is prohibited without appropriate approvals and materials specifically manufactured for such use.
For formulation researchers conducting preclinical method development using research‑grade materials, independently verify purity, sequence, and endotoxin, and transition to appropriate GMP‑compliant supply chains before regulated studies.
Physical Properties
Item-specific physico-chemical specifications were not provided for this siRNA set.
Appearance/State: Not specified for this item; refer to CoA/Spec Sheet. siRNAs are commonly supplied lyophilized or in aqueous RNase‑free buffer (general, literature).
Molecular weight: Not specified for this item; depends on sequence and length.
Solubility (general, literature):
Freely soluble in nuclease-free water or aqueous buffers (e.g., 10 mM Tris, 1 mM EDTA, pH 7–8).
Insoluble in nonpolar organic solvents.
pH stability (general, literature): Stable in near‑neutral buffers (pH ~6.5–8.0). Avoid strong acid/base to prevent depurination/degradation.
UV characteristics (general, literature): siRNA concentration can be estimated at 260 nm; A260 of 1.0 corresponds to ~40 µg/mL for single-stranded RNA; for duplex, use vendor-provided extinction coefficient when available. Not specified for this item; refer to CoA/Spec Sheet.
Endotoxin/bioburden/metal content/UV cutoffs: Not specified for this item; refer to CoA/Spec Sheet.
Practical notes (general)
Reconstitution is typically performed at 10–100 µM in RNase‑free water or buffer; vortex gently and spin down to collect solution.
Store reconstituted aliquots at −20 °C or below; avoid repeated freeze–thaw cycles.
All values above labeled “general” are literature guidance only and are not product specifications.
Quality and Grades
Item-specific grade/purity was not provided.
Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
Context for siRNA quality (general, literature):
Desalted vs. HPLC‑purified: Desalted siRNA removes small-molecule impurities/salts but may retain synthesis truncations. HPLC purification reduces truncated species, typically improving knockdown performance and consistency.
PAGE purification: Offers stringent size-based purification at the expense of yield; sometimes used for sensitive applications.
Modifications: 2′‑O‑Me, 2′‑F, phosphorothioate, or terminal conjugations can enhance stability/uptake; presence/absence not specified for this item.
QC metrics typically reported: OD260, duplex concentration, purity by HPLC/PAGE, residual solvents/salts, endotoxin (EU/mg), bioburden, and identity by mass spectrometry. For this item, these metrics are not specified; refer to CoA/Spec Sheet.
Implications for experimental use
If grade is unspecified, validate performance by a small pilot transfection and quantify knockdown by RT‑qPCR or immunoblot.
For sensitive in vivo‑like models or primary cells, higher-purity (HPLC/PAGE) and low-endotoxin materials are often preferred (general guidance).
Documentation
For definitive acceptance criteria (purity %, residual salts/solvents, endotoxin limits), consult the item’s Certificate of Analysis and Specification Sheet.
Reaction and Applications
Primary application: sequence‑specific knockdown of mouse Ddr1 mRNA via the RNA interference (RNAi) pathway in cultured cells or other in vitro systems.
Mechanistic overview (general, literature):
Duplex siRNA is recognized by RISC; the antisense (guide) strand directs Argonaute‑mediated cleavage of complementary Ddr1 mRNA, reducing transcript levels and subsequent protein expression.
Typical experimental use cases:
Pathway interrogation: Assess collagen‑mediated signaling and epithelial/mesenchymal processes modulated by Ddr1.
Target validation: Correlate Ddr1 knockdown with phenotypic readouts in cell models.
Combination perturbations: Co‑transfect with siRNAs to non-overlapping targets or with rescue plasmids encoding siRNA‑resistant Ddr1 to confirm on-target effects.
Controls (general):
Non‑targeting siRNA control and positive control siRNA (e.g., targeting a housekeeping gene with known knockdown) to benchmark transfection efficiency.
Off‑target assessment via multiple independent duplexes; use pooled duplexes to mitigate single‑site off‑targets.
Practical tips:
Optimize siRNA dose (final 1–50 nM, commonly 5–20 nM) and transfection reagent ratio per cell type.
Quantify knockdown at 24–72 h by RT‑qPCR; protein changes may require 48–96 h depending on protein half‑life.
Use low‑passage cells and confirm mycoplasma‑free status to reduce variability.
Item-specific validation data or recommended conditions: Not specified for this item; consult CoA/Spec Sheet or internal application notes if available.
Reaction Conditions
While not a chemical reaction reagent, siRNA performance depends on transfection and culture conditions. The following are general, literature‑based guidelines to help plan experiments; they are not item‑specific specifications.
Cell density at transfection: 30–60% confluence (adherent lines) to ensure active proliferation and uptake.
Dose range: Final 1–50 nM siRNA; common starting points 5–10 nM for robust cell lines, 10–25 nM for primary/fragile cells. Titrate to minimize off‑target effects.
Complexation: Mix siRNA with lipid/polymer transfection reagent in serum‑free medium (e.g., Opti‑MEM). Incubate 10–20 minutes at room temperature to form complexes, then add dropwise to cells.
Medium: Replace after 4–24 h if cytotoxicity is observed; many protocols keep complexes for full duration if tolerated.
Incubation time course: mRNA nadir often at 24–48 h; protein knockdown at 48–96 h depending on turnover. Plan endpoint assays accordingly.
Controls: Include non‑targeting siRNA, mock transfection, and (optionally) a positive control siRNA to benchmark delivery efficiency.
Readouts: RT‑qPCR for Ddr1 mRNA; Western blot for DDR1 protein and phospho‑DDR1 following collagen stimulation; pathway markers (e.g., p‑ERK) as functional correlates.
Co‑treatments: For DDR1 pathway studies, collagen I/IV stimulation post‑transfection can elucidate functional impacts of knockdown.
Item‑specific, validated conditions or transfection reagent recommendations: Not specified for this item; refer to CoA/Spec Sheet.
Safety and Handling
Safety classifications were not supplied for this item.
GHS classification, pictograms, signal word, and H-statements: Not specified for this item; consult the product SDS for authoritative information.
Research use restriction: For research use only (as provided).
General laboratory safety guidance for RNA oligonucleotides (literature):
Hazard profile: Synthetic siRNAs are generally considered low hazard. Avoid inhalation of lyophilized powders and contact with eyes/skin.
Personal protective equipment: Lab coat, gloves, and safety glasses. Use RNase-free technique to prevent degradation and potential contamination of workspaces.
Handling: Work in a clean area dedicated to RNA work. Use certified RNase‑free consumables. Change gloves frequently; avoid touching common surfaces with gloved hands during RNA handling.
Incompatibilities: Avoid strong oxidants and bases/acids that can degrade RNA. Keep solutions free of divalent RNases cofactors (e.g., Mg2+) unless required for downstream enzymology.
First aid (general): If inhaled, move to fresh air. In case of skin or eye contact, rinse with water for at least 15 minutes. If ingested, rinse mouth with water. Seek medical attention if irritation persists. Follow institutional protocols.
Spills and disposal: Absorb aqueous spills with appropriate materials and decontaminate with RNase-inactivating solutions or 10% bleach, followed by water. Dispose according to local regulations and institutional biosafety rules.
Always defer to the SDS and your institutional EHS guidance for definitive safety instructions.
Solvent Selection
As an RNA duplex reagent, this product is formulated and used in aqueous systems rather than organic solvents.
Primary solvents/buffers (general, literature):
Nuclease-free water (RNase/DNase‑free) for initial reconstitution.
TE or TE‑Low buffer (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0) to chelate trace divalent cations and enhance stability.
For cell delivery, dilute in serum‑containing or serum‑free culture medium immediately before complexation with a transfection reagent.
Miscibility: Fully miscible with aqueous media. Avoid organic solvents which can denature oligonucleotides and are generally incompatible with cells.
Dielectric/polarity: High-polarity aqueous systems are standard for nucleic acids (general).
When to choose specific media (general guidance)
Serum‑free complexation often improves lipoplex formation; after 10–30 minutes complexation, add to complete medium.
Presence of Mg2+/Ca2+ can promote RNase activity; buffering with EDTA during storage (not during transfection) can help stability.
Comparison (general)
Water vs TE: Water avoids introducing salts to sensitive cells; TE provides better storage stability. Many users store in TE‑Low and dilute into water/medium for transfection.
Item-specific solvent recommendations: Not specified for this item; refer to CoA/Spec Sheet.
General guidance for siRNA handling (literature; supplementing the above)
Upon receipt: Inspect packaging integrity. If lyophilized, allow contents to equilibrate to room temperature before opening to prevent condensation. If in solution, keep on ice during setup.
Reconstitution:
Solvent: RNase‑free water or TE‑Low (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH 7.5–8.0).
Concentration: Prepare stocks at 10–100 µM; 20 µM is a common working stock. Mix gently by pipetting; avoid vigorous vortexing that can shear nucleic acids.
Annealing: Most commercial siRNAs are pre‑annealed; no further annealing is required unless specified. Not specified for this item; refer to CoA/Spec Sheet.
Aliquoting: Dispense small, single‑use aliquots (e.g., 5–20 µL) in RNase‑free tubes to minimize freeze–thaw.
Storage of reconstituted stocks: −20 °C (weeks–months) or −80 °C (months–year). Avoid >3 freeze–thaw cycles.
Working solutions: Keep on ice during use; discard leftover diluted complexes after transfection—do not refreeze.
Light sensitivity: siRNA without chromophores is not strongly light‑sensitive; nonetheless, store protected from light to minimize potential degradation.
Item-specific details such as exact solvent, concentration, stabilizers, or shelf life: Not specified for this item; refer to CoA/Spec Sheet.
Always adhere to RNase‑free technique to preserve integrity and performance.
Structure and Identity
This product is a pre-designed small interfering RNA (siRNA) pool targeting mouse Ddr1 (Discoidin domain receptor 1). As an siRNA reagent, it consists of short double‑stranded RNAs intended for RNAi-mediated knockdown of Ddr1 transcripts.
SKU: D1460146
Product type: Pre-designed siRNA Set A (mouse Ddr1), typically a set of multiple duplexes targeting distinct sites (general for “Set A” offerings; exact count/sequences not specified 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.
SMILES / InChI / InChIKey: Not applicable to heterogeneous oligonucleotide mixtures; not specified for this item.
Structural features (general, literature):
Double-stranded RNA duplexes ~19–21 bp with 2-nt 3′ overhangs are typical for synthetic siRNA designs.
Sense strand is complementary to target mRNA; antisense (guide) strand loads into RISC.
Phosphate backbone with ribose sugars; optional chemical stabilizations may be present in some products (not specified for this item).
2D description (general): Two antiparallel RNA strands aligned in an A-form duplex with terminal overhangs; bases paired via Watson–Crick complementarity.
Any item-specific sequence, length, chemical modifications, or strand stoichiometry: Not specified for this item; refer to CoA/Spec Sheet.
Synthetic Utility
Not typically applicable. This product is a functional, sequence‑defined oligonucleotide reagent for gene knockdown rather than a small‑molecule building block.
General notes (literature):
Chemical synthesis: siRNAs are produced via solid‑phase phosphoramidite chemistry with iterative coupling/oxidation/deprotection and post‑synthetic annealing. These processes occur at the manufacturer and are not intended as user‑facing synthetic steps.
Derivatization: Researchers sometimes incorporate 2′‑O‑Me, 2′‑F, PS linkages, or terminal conjugates to modulate stability and delivery; presence/absence for this item is not specified.
For experimental planning, the relevant sections are Reaction & Applications and Application Protocols, which cover how to use this siRNA in cell‑based assays rather than any role as a synthetic intermediate.
Species reactivity: Mus musculus (mouse), based on product name.
Format: Pre‑designed siRNA Set A, typically a pool of distinct duplexes targeting non‑overlapping regions of the Ddr1 transcript (general for pooled siRNA sets; exact sequences or target regions are not specified for this item).
Off‑target minimization (general, literature): Pooled duplexes reduce the concentration of any single sequence, mitigating off‑target effects while maintaining on‑target silencing. BLAST screening and seed region analysis are standard in design pipelines.
Item‑specific details
Guide/sense sequences, target exon positions, seed regions, and chemical modifications: Not specified for this item; refer to CoA/Spec Sheet.
Verified cross‑reactivity to related kinases or DDR isoforms: Not specified for this item.
Recommended verification (general)
Confirm on‑target action with multiple duplexes, dose‑response, and rescue with siRNA‑resistant Ddr1 cDNA.
Monitor potential seed‑mediated off‑targets by transcriptome profiling where critical.
Perguntas frequentes
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.
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