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| Targets |
This negative control targets the asialoglycoprotein receptor (ASGPR) on hepatocytes through its GalNAc (N-acetylgalactosamine) ligand. ASGPR is a C-type lectin highly expressed on the sinusoidal surface of hepatocytes. The GalNAc ligand binds specifically and with high affinity to ASGPR, leading to rapid internalization of the siRNA conjugate via endocytosis. However, because the siRNA sequence is scrambled, it does not bind to the target mRNA (e.g., TTR) within the cell. Thus, it serves as an ideal control to ensure that any effects seen with the active GalNac-siRNA are due to specific mRNA sequence recognition and not due to ASGPR delivery or the siRNA backbone. It functions within the PROTAC/LYTAC pathway.
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| ln Vitro |
In vitro, this negative control is used in cell-based assays to validate target gene knockdown specificity. It should not knock down the intended target mRNA (e.g., TTR). A typical protocol: Primary human hepatocytes or HepG2 cells (which express ASGPR) are seeded in 12-well plates. The cells are treated with 1-100 nM of the GalNac-siRNA negative control (21nt) for 24-96 hours. The knockdown of the target gene (e.g., TTR) is measured by qRT-PCR and Western blot. The control should show no significant reduction in target gene expression compared to untreated cells, whereas the active GalNac-siRNA should show potent knockdown. This confirms that the activity of the active siRNA is sequence-specific.
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| ln Vivo |
In vivo, GalNac-siRNA negative control (21nt) is administered to animal models to control for non-specific pharmacology. A typical study: Male C57BL/6 mice (n=8/group) are injected subcutaneously (SC) with a single dose (e.g., 1-10 mg/kg) of the negative control formulation. Blood is collected at regular intervals (e.g., days 7, 14, 21, and 28). The levels of the target protein (e.g., TTR) in the plasma are measured by ELISA. The negative control should not alter the plasma concentration of the target protein compared to the vehicle control group. This confirms that the therapeutic effect (e.g., reduction in TTR) of the active GalNac-siRNA is due to its specific RNAi sequence.
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| Enzyme Assay |
For non-cellular assays, GalNac-siRNA negative control (21nt) is used as a control in binding studies to assess ASGPR specificity. A surface plasmon resonance (SPR) assay can be used. Immobilize recombinant human ASGPR on a sensor chip. Flow the GalNac-siRNA negative control (21nt) over the chip at various concentrations (e.g., 1-1000 nM) in running buffer. The binding affinity (KD) can be calculated based on the association and dissociation rate constants. The negative control should bind ASGPR with similar affinity as the active GalNac-siRNA, as the ASGPR binding is conferred by the GalNAc ligand, not the siRNA sequence. This validates that the targeting ligand is functional.
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| Cell Assay |
For in vitro cell assays, a typical protocol: Human hepatoma cells (Huh-7) are seeded in 96-well plates at 10,000 cells/well. After 24 hours, cells are treated with GalNac-siRNA negative control (21nt) at concentrations ranging from 1 nM to 1 uM. The compound is dissolved in sterile nuclease-free water to a stock solution (e.g., 20 uM) and added directly to the culture medium. Transfection reagents are typically not required as the GalNAc mediates efficient ASGPR uptake. After 72 hours, cells are lysed. RNA is extracted and the expression of a panel of off-target genes (e.g., inflammatory cytokines) is measured by qRT-PCR. The negative control should induce minimal off-target gene expression compared to the untreated control.
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| Animal Protocol |
For in vivo animal studies, GalNac-siRNA negative control (21nt) is a critical tool. A typical study in mice (e.g., C57BL/6) bearing a human TTR transgene: Mice are dosed subcutaneously (SC) with the control at 1 mg/kg, 3 mg/kg, and 10 mg/kg (n=5 per group). Dosing may be single or multiple (e.g., weekly for 4 weeks). Blood samples are collected from the tail vein pre-dose and at various timepoints post-dose (e.g., days 7, 14, 21, 28). Plasma is separated, and the concentration of the target protein (e.g., human TTR) is measured by a specific ELISA. The negative control group should maintain stable plasma TTR levels, indistinguishable from the vehicle (PBS) control group, validating the specificity of the active GalNac-siRNA.
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| ADME/Pharmacokinetics |
Pharmacokinetics: As a GalNAc-conjugated siRNA, the negative control is expected to have identical PK/ADME properties to the active therapeutic siRNA, as the GalNAc ligand drives the biodistribution. It is highly stable in plasma and primarily taken up by the liver via ASGPR-mediated endocytosis. After subcutaneous or intravenous administration, it is rapidly distributed to the liver. The plasma half-life (t½) is relatively short (hours), but tissue residence is long (weeks). It accumulates in the liver for extended periods, allowing for durable pharmacodynamic effects. The negative control is used to control for these non-specific distribution and accumulation effects. Metabolism occurs primarily within the endosomes/lysosomes.
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| Toxicity/Toxicokinetics |
Toxicity: GalNac-siRNA negative control (21nt) is used to control for non-specific toxicities of the active drug. It should exhibit a similar toxicity profile to the active, but without target-mediated side effects. In animal studies, it is generally well-tolerated at therapeutic doses. Toxicities may include injection site reactions, mild thrombocytopenia, and complement activation. Off-target RNAi effects or immune stimulation (cytokine release) can occur at high doses; the negative control is used to identify such effects. Standard safety precautions: avoid inhalation and ingestion. Use PPE. Not for human use. For research use only.
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| References | |
| Additional Infomation |
Other information: GalNac-siRNA negative control (21nt). Appearance: Solid, White to off-white. Storage: -20degC, sealed, away from moisture; In solvent: -80degC for 6 months, -20degC for 1 month. Shipping: room temperature. For research use only-not for human use. Targets: ASGPR (via ligand). Sequence length: 21 nucleotides. Used as a negative control for RNAi experiments in hepatocytes.
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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