GPAM Human Pre-designed siRNA Set A

Cat. No.: G1479679

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Visão geral

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

Condições de armazenamento de armazenamento

Store at -20°C

Enviado em

Ice chest + Ice pads

Este produto requer transporte de cadeia fria. Serviços terrestres e outros serviços econômicos não estão disponíveis.

Product Line

Produtos biológicos

Application Protocols

No item‑specific, vendor‑validated protocols are provided in the product data. The following is a general workflow for transfecting siRNA into human cell lines to knock down GPAM (literature/general):

• Preparation:
  • Thaw siRNA on ice; briefly spin. Work RNase‑free. Reconstitute to 20–50 µM in RNase‑free water or TE; make single‑use aliquots.
• Complex formation (example lipid‑mediated protocol; follow your reagent’s IFU):
  • Dilute siRNA in serum‑free medium (e.g., Opti‑MEM) to 2× the desired final concentration (e.g., 10–100 nM).
  • Separately dilute transfection reagent per manufacturer’s recommendation. Incubate reagent and siRNA together for 10–20 min at room temperature to form complexes.
• Transfection:
  • Seed cells to reach 50–70% confluence at transfection. Replace with fresh serum‑free or low‑serum medium if required. Add complexes dropwise with gentle rocking.
  • Incubate 4–6 h (or as directed), then replace with complete growth medium. Some reagents permit no‑change protocols.
• Post‑transfection:
  • Harvest RNA at 24–48 h for RT‑qPCR; harvest protein at 48–96 h for Western blot or activity assays.
  • Include controls: non‑targeting siRNA, mock transfection, and if possible, a positive control siRNA with known phenotype.
• Troubleshooting tips:
  • If viability drops, lower siRNA dose and/or reagent amount. If knockdown is weak, test multiple doses (5–50 nM), alter cell density, or switch reagent.

Scale volumes according to plate format; maintain aseptic technique and RNase control.

Biological Roles

This siRNA set targets human GPAM (glycerol‑3‑phosphate acyltransferase, mitochondrial), a key enzyme in glycerolipid biosynthesis. The following summarizes GPAM biology (literature/general):

• Enzymatic function: GPAM catalyzes the acylation of glycerol‑3‑phosphate to lysophosphatidic acid (LPA), the committed first step in de novo triacylglycerol and phospholipid synthesis. It typically prefers saturated acyl‑CoA substrates and operates on the outer mitochondrial membrane.
• Pathway context: LPA produced by GPAM is further acylated to phosphatidic acid and then converted to diacylglycerol and triacylglycerol or to glycerophospholipids. GPAM thus influences cellular lipid droplet formation and membrane lipid composition.
• Regulation: Expression and activity are modulated by nutritional and hormonal cues affecting lipid metabolism; transcriptional control involves lipid‑sensitive pathways. Post‑translational mechanisms and substrate availability also contribute.
• Functional studies: RNAi‑mediated knockdown of GPAM is commonly used to probe lipid flux, glycerolipid remodeling, and metabolic coupling to mitochondrial function in diverse human cell systems.
• Redundancy and specificity: Mammalian cells express multiple glycerol‑3‑phosphate acyltransferases (e.g., GPAT isoforms). Targeted silencing of GPAM helps disentangle mitochondrial GPAT‑specific contributions from endoplasmic reticulum isoforms.

Note: The above biological context is provided to assist experimental design and interpretation. This product is for laboratory research use only; no diagnostic or therapeutic claims are made or implied.

Buffer Applications

While not a classical buffering reagent, successful siRNA use depends on appropriate reconstitution and storage buffers. The guidance below is literature/general and should be tailored to your workflow; item‑specific formulations are not specified.

• Reconstitution options:
  • RNase‑free water (nuclease‑free): Ideal for maximal compatibility with downstream enzymatic steps; prepare concentrated stocks (e.g., 20–50 µM).
  • TE or TE‑low (10 mM Tris·HCl, pH 7.5–8.0; 0.1–1 mM EDTA): Enhances long‑term stability via metal chelation; verify that EDTA will not interfere with Mg2+‑dependent assays.
• Working buffers:
  • For transfection complexation, follow the transfection reagent manufacturer’s buffer system (often proprietary dilution buffers or Opti‑MEM‑like reduced‑serum media). Avoid serum during complex formation unless the reagent permits it.
• Storage considerations:
  • Salt and divalent cations can promote nuclease activity if contamination occurs; maintain low ionic strength for storage. Filter sterilize (0.22 µm) if sterility is required.
• Practical recipes (examples):
  • 1× TE: 10 mM Tris·HCl, 1 mM EDTA, pH 7.5–8.0 using RNase‑free components.
  • TE‑low: 10 mM Tris·HCl, 0.1 mM EDTA, pH 7.5–8.0.

Do not use this product as a pH buffer in analytical chemistry. Buffer selection here is strictly for siRNA stability and handling.

Green Alternatives

For siRNA workflows, environmental impact is largely governed by solvent/buffer choices, plastics usage, and cold‑chain logistics rather than the active reagent. The following considerations (literature/general) can reduce footprint without compromising data quality:

• Prefer aqueous systems: Use RNase‑free water or minimal‑component Tris buffers. Avoid unnecessary organic solvents during handling; reserve alcohols for precipitation steps only when required.
• Minimize single‑use plastics: Where compatible with RNase control, adopt RNase‑free reusable racks and reduce overpackaging. Select low‑binding tubes to lower repeat consumable use.
• Concentrated stocks: Reconstitute at higher concentrations (e.g., 20–50 µM) to reduce storage volume and packaging; aliquot to right‑size working volumes and minimize waste from freeze–thaw cycles.
• Energy savings: Consolidate shipments and align order timing to reduce repeated cold‑chain transports. Store long‑term at −20 °C or −80 °C as appropriate; use frost‑free, energy‑efficient freezers with proper maintenance.
• Low‑toxicity transfection: Choose delivery reagents with favorable environmental and cytotoxicity profiles; newer lipid formulations can achieve potency at reduced doses, decreasing reagent consumption and waste.

Comparison (illustrative):

• Aqueous TE vs buffered saline with preservatives: TE avoids added biocides, leverages chelation for stability, and simplifies disposal.
• Electroporation vs organic solvent–assisted delivery: Electroporation eliminates solvent waste but may increase electrical energy use; balance based on scale and cell type.

Item‑specific green attributes (e.g., solvent‑free formulation, certifications): Not specified for this item.

Pharmaceutical Uses

This product is supplied strictly for research use only and is not intended for human or veterinary use, diagnostics, or clinical applications. No pharmacopeial designation is provided for this item.

Relevant formulation/CMC considerations for siRNA in a research and development context (literature/general; not claims for this specific item):

• Excipient compatibility: siRNAs interact with cationic lipids/polymers to form nanoparticles; buffer composition (pH ~7–8, low ionic strength) influences complexation, size, and zeta potential.
• Sterility/endotoxin: For parenteral‑style research models, low endotoxin and sterile filtration (0.22 µm) are often pursued; specifications for this item are not provided.
• Stability: Chemical modifications and lyophilization with stabilizers (e.g., trehalose, sucrose) can enhance stability during storage and shipping; the presence of such excipients is not specified for this item.
• Analytical methods: A260 UV, ion‑pair HPLC/UPLC, capillary gel electrophoresis, and mass spectrometry are commonly used to characterize oligonucleotide integrity and purity in development settings.

Again, none of the above constitutes approval or suitability for therapeutic use. For manufacturing or preclinical development, consult internal quality systems and regulatory guidance; this catalog item provides a research‑grade tool for target validation and mechanism studies.

Physical Properties

Item-specific physicochemical specifications are not provided for this siRNA set; consult the CoA/Spec Sheet for exact details. The following properties reflect typical siRNA behavior and are provided as literature/general guidance for planning:

• Physical state: Often supplied lyophilized or in aqueous buffer; appearance for this item is not specified.
• Solubility (literature/general): Freely soluble in RNase‑free water or aqueous buffers (e.g., 10 mM Tris·HCl, pH 7.5–8.0; TE). Poorly soluble in most organic solvents.
• pH stability (literature/general): Stable near neutral pH; avoid strong acid/base which can hydrolyze the phosphodiester backbone or depurinate nucleobases.
• Thermal behavior (literature/general): Duplex melting temperature (Tm) depends strongly on sequence/length and salt; typical 19–21‑mer siRNAs exhibit Tm values ~60–85 °C in physiological ionic strength, but exact Tm is sequence‑dependent and not specified for this item.
• UV absorbance (literature/general): Strong A260 absorbance; ε depends on sequence. Concentration can be estimated using A260 (1 A260 ≈ 40 µg/mL for single‑stranded RNA; duplex values differ slightly). Not specified for this item; verify with CoA if provided.
• Molecular weight (item-specific): Not specified for this item; refer to CoA/Spec Sheet.
• Additives/stabilizers: Not specified for this item; refer to CoA/Spec Sheet.

Note: Handle under RNase‑free conditions to preserve integrity; avoid repeated freeze–thaw which accelerates strand cleavage and aggregation.

Quality and Grades

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

Context for siRNA reagent quality (literature/general):

• Oligonucleotide quality dimensions commonly include coupling efficiency, overall purity (% full‑length), sequence identity, and endotoxin/bioburden testing. Purification methods may include cartridge desalting, PAGE, or HPLC; higher‑stringency methods (e.g., HPLC) typically reduce truncated species and improve knockdown consistency.
• Quantitation is typically by A260 with sequence‑corrected extinction coefficients. Mass confirmation (e.g., MALDI‑TOF or ESI‑MS) is often used to verify average molecular mass; presence/absence of such testing is not specified for this item.
• Duplexing quality: For pre‑annealed duplexes, correct equimolar strand assembly and low single‑strand carryover are important; annealing conditions and verification (e.g., native PAGE) may be documented on the CoA.
• Stabilization: Some siRNA products include limited 2′‑modifications or terminal phosphorothioates to enhance nuclease resistance and in‑cell potency; whether such modifications are present is not specified for this item.
• Functional performance: Vendors frequently benchmark pools/sets against mRNA knockdown by qPCR/RT‑qPCR or protein reduction by Western blot in standard cell lines. Performance claims and acceptance criteria are not specified for this item; consult the product documentation.

Recommendation: Review the CoA/Spec Sheet for this lot’s purification method, quantitation, residual salts/solvents, and any sterility/endotoxin data before critical experiments.

Reaction and Applications

As a gene‑silencing reagent, this siRNA set is designed for sequence‑specific knockdown of human GPAM transcripts via the RNA interference (RNAi) pathway. While not used in classical synthetic “reactions,” the following application domains are typical for siRNA (literature/general):

• Cell culture knockdown: Transient suppression of GPAM expression in human cell lines using lipid‑mediated transfection, electroporation, or polymeric delivery systems. Typical working concentrations are 5–50 nM siRNA, optimized per cell type and transfection reagent.
• Mechanism: The antisense strand loads into RISC, guiding Argonaute‑mediated endonucleolytic cleavage or translational repression of complementary GPAM mRNA.
• Experimental readouts: qPCR/RT‑qPCR for mRNA reduction; Western blot, ELISA, or targeted proteomics for protein decrease; functional assays of glycerolipid biosynthesis or acyltransferase activity to confirm pathway impact.
• Pooled/Set strategy: Using multiple distinct siRNA designs against GPAM helps mitigate off‑target artifacts and sequence‑specific idiosyncrasies. Pooling at equal molarity or rotating individual duplexes are common practices.
• Controls: Non‑targeting siRNA, transfection reagent only, and positive control siRNA (against a housekeeping gene with measurable phenotype) strengthen interpretation.
• Co‑transfections: Combine with reporter constructs, rescue plasmids (siRNA‑resistant GPAM cDNA), or CRISPR tools to dissect on‑target effects.

Item‑specific application validation, tested cell lines, and expected knockdown levels: Not specified for this item; consult the product documentation and optimize conditions empirically.

Reaction Conditions

Classical chemical reaction conditions are not applicable to siRNA. Instead, experimental conditions relevant to successful GPAM knockdown are summarized below as literature/general guidance. Item‑specific validated parameters are not provided.

• Transfection conditions:
  • Cell confluence: Commonly 50–70% at the time of transfection for adherent lines; optimize per cell type.
  • siRNA dose: Typical screening range 5–50 nM final; higher doses can increase off‑target effects and cytotoxicity.
  • Complexation: Follow the transfection reagent’s protocol (incubation time, buffer, N/P ratio). Prepare complexes in serum‑free medium unless the reagent allows serum.
  • Incubation: 4–6 h complex exposure followed by medium change is common; some reagents support no‑change protocols.
• Timelines:
  • mRNA reduction: Often detectable within 24 h, maximal by 24–72 h.
  • Protein reduction: Depends on target protein half‑life; typically 48–96 h.
• Controls and replication:
  • Include non‑targeting siRNA, mock transfection, and if possible, a GPAM siRNA‑resistant rescue construct to confirm on‑target effects.
• Data quality:
  • Assess cell health (viability assays) to exclude toxicity‑driven phenotypes.
  • Quantify knockdown by RT‑qPCR (normalized to stable reference genes) and protein assays.

Environmental parameters (temperature, CO2): Standard mammalian culture conditions (e.g., 37 °C, 5% CO2) unless your cell model requires otherwise.

Safety and Handling

• Regulatory status: For research use only (not for diagnostic, therapeutic, or in vivo clinical applications).
• GHS classification, signal word, pictograms, H‑statements: Not specified for this item; refer to the SDS for authoritative safety information.

General laboratory precautions for RNA reagents (literature/general):

• Personal protective equipment: Wear lab coat, nitrile gloves, and safety glasses. Change gloves frequently to minimize RNase contamination.
• RNase control: Use RNase‑free consumables and reagents; treat work surfaces and tools with RNase decontamination solution. Work quickly on ice during setup to limit nuclease activity.
• Incompatibilities: Avoid strong acids/bases and oxidizing agents that degrade RNA. Do not mix with RNase‑containing materials (e.g., non‑sterile biological fluids) unless part of an experimental protocol.
• First aid (general): In case of contact with eyes/skin, rinse with water. If inhaled or ingested (unlikely in typical handling), seek medical advice and show the SDS.
• Spill response: Wipe with RNase‑decontaminating solution, then with water/ethanol; dispose of waste according to institutional guidelines for nucleic acid reagents.
• Waste: Aqueous buffers containing RNA can generally be disposed as non‑hazardous chemical waste unless mixed with hazardous components; follow local regulations.

Shipping and storage conditions provided for this item: Shipped in an ice chest with ice pads; store at −20 °C. Protect from repeated freeze–thaw by aliquoting upon first thaw.

Solvent Selection

This product is an aqueous‑compatible nucleic acid reagent; conventional organic solvent selection paradigms do not apply. The following guidance reflects typical handling of siRNA (literature/general):

• Primary solvents: RNase‑free water, 1× or 0.5× TE (10 mM Tris·HCl, 0.1–1 mM EDTA, pH 7.5–8.0). Low‑salt Tris buffers help maintain integrity and reduce metal‑catalyzed nuclease activity.
• Avoid: Strongly acidic/basic solutions; divalent cation‑rich buffers (e.g., high Mg2+/Ca2+) can promote nuclease activity in contaminated environments. High concentrations of organic cosolvents can destabilize duplexes.
• Cosolvents (as needed): Up to ~10–20% sterile glycerol may be used in some workflows for freeze–thaw resilience; ethanol or isopropanol is used for precipitation/purification but not for storage.
• pH: Maintain near neutral (pH 7–8). Slightly alkaline Tris buffers are standard to minimize acid‑catalyzed hydrolysis.
• Filtration: If sterile solutions are required, use 0.22 µm low‑binding filters; avoid shear and RNase introduction.

Comparison (literature/general):

• Water vs TE: Water is simplest and avoids EDTA interference in Mg2+‑dependent downstream enzymes; TE offers improved long‑term stability by chelating trace metals.
• PBS: Physiological saline buffers are generally acceptable for short‑term use but may not be ideal for long‑term storage due to ionic strength and potential nuclease contamination.

Item‑specific solvent recommendations (exact compositions, concentrations): Not specified for this item; refer to CoA/Spec Sheet.

Storage and Reconstitution

• Storage conditions (item‑specific): Store at −20 °C. Shipped in an ice chest with ice pads.
• Form/appearance: Not specified for this item; refer to CoA/Spec Sheet (may be lyophilized or in solution).

General guidance for siRNA handling (literature/general):

• Reconstitution:
  • Use RNase‑free water or TE (10 mM Tris·HCl, 0.1–1 mM EDTA, pH 7.5–8.0).
  • Prepare a concentrated stock (e.g., 20–50 µM). Vortex gently and brief spin; allow 10 min at room temperature to fully dissolve.
  • If supplied single‑stranded, anneal per vendor instructions; if pre‑duplexed, proceed directly to aliquoting.
• Aliquoting and stability:
  • Dispense into single‑use, low‑binding RNase‑free tubes to avoid repeated freeze–thaw. Typical aliquots: 5–20 µL.
  • Short‑term storage: 2–8 °C for up to 1 week is common; verify stability empirically.
  • Long‑term storage: −20 °C (as specified) or −80 °C for extended duration. Avoid frost‑free freezers that cycle temperatures.
  • Protect from light only if fluorescently labeled (not specified for this item).
• Thawing/handling:
  • Thaw on ice, mix gently (avoid foaming), quick spin to collect. Keep on ice during setup.
• Documentation:
  • Record lot number, reconstitution buffer, concentration, and aliquot map. Retain CoA and SDS for compliance.

Exact concentration, yield, and any stabilizers or preservatives: Not specified for this item; refer to CoA/Spec Sheet.

Structure and Identity

This product is a pre-designed small interfering RNA (siRNA) reagent set targeting the human GPAM gene (glycerol-3-phosphate acyltransferase, mitochondrial; often referred to as GPAT1). Each siRNA is a short, double‑stranded RNA duplex intended to guide RISC‑mediated silencing of GPAM mRNA.

• Item-specific identifiers:

  • SKU: G1479679
  • Product name: GPAM Human Pre-designed siRNA Set A
  • CAS, CID, InChIKey, SMILES: Not applicable to siRNA mixtures; not specified for this item.
  • Molecular formula and exact molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

• Structural features (literature/general):

  • Typical siRNA duplexes are 19–21 bp with 2‑nt 3′ overhangs, phosphate diester backbones, and ribose sugars bearing 2′‑OH groups.
  • May include sense and antisense strands with sequence complementarity to a region of human GPAM mRNA; antisense (guide) strand loads into RISC.
  • Some commercial siRNAs incorporate chemical stabilizations (e.g., 2′‑OMe, 2′‑F, phosphorothioate) in limited positions to enhance stability; presence/positions are not specified for this item.

• 2D description (literature/general):

  • Repeating ribonucleotide units (A, U, G, C) linked via 3′–5′ phosphodiester bonds, forming two antiparallel strands annealed by Watson–Crick base pairing (A:U, G:C) with short unpaired 3′ overhangs.

• Target identity (general):

  • Gene: GPAM (human); mitochondrial acyltransferase catalyzing the first committed step of glycerolipid biosynthesis. Specific target region(s) and sequences are not provided in the product data.

Synthetic Utility

Not typically applicable. This product is a biological reagent (siRNA duplexes) rather than a small‑molecule building block or synthetic intermediate.

For researchers integrating siRNA with chemical biology (literature/general):

• Conjugation chemistry: siRNAs can be modified post‑synthesis with functional groups (e.g., amines, thiols, azides) enabling click chemistry (CuAAC, SPAAC) to attach reporters, lipids, or ligands. Such modifications, if present, would be defined in the product documentation; none are specified for this item.
• Cross‑disciplinary workflows: Small‑molecule inhibitors or activators of lipid metabolism can be combined with GPAM knockdown to dissect pathway control, but the siRNA itself is not used as a reagent in organic reactions.

Item‑specific functional groups, protecting groups, and reactivity: Not specified for this item; refer to CoA/Spec Sheet if chemically modified variants are offered.

Target Specificity

• Intended target: Human GPAM mRNA (glycerol‑3‑phosphate acyltransferase, mitochondrial), commonly referred to as GPAT1.
• Species: Homo sapiens.
• Modality: RNA interference (siRNA duplexes) designed to achieve sequence‑specific post‑transcriptional gene silencing via RISC loading of the antisense guide strand.
• Design approach (literature/general): Pre‑designed siRNA sets typically comprise multiple distinct duplexes targeting non‑overlapping regions of the transcript to enhance robustness and mitigate off‑target artifacts. Exact sequence(s), target exon(s), and strand orientation are not provided for this item.
• Off‑target considerations (literature/general):
  • Seed‑region complementarity (positions 2–8 of the guide) can downregulate unintended transcripts; using pools at lower per‑duplex concentrations and validating phenotypes with independent sequences reduce risk.
  • Immune activation is minimized by rational design and, where used, selective chemical modifications; the presence of such features is not specified for this item.
• Verification strategies:
  • Confirm on‑target activity by RT‑qPCR and Western blot; perform rescue with siRNA‑resistant GPAM cDNA to demonstrate specificity.

Item‑specific target regions, sequences, predicted off‑target lists, and in silico design scores: Not specified for this item; consult the CoA/Spec Sheet or design documentation if provided.

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.