Rat Paqr3 Pre-designed siRNA

Overview

This pre-designed siRNA set enables sequence-specific knockdown of the rat Paqr3 gene (NCBI Gene ID: 305203) in rat cell lines. Each set is supplied as HPLC-purified, chemically synthesized double-stranded RNA oligonucleotides in lyophilized form, ready for reconstitution and transfection. Two size formats are available: Set A (SI210050A, 3 siRNA sequences at 3×5 nmol) and Set B (SI210050B, 4 siRNA sequences at 4×10 nmol), providing flexibility for screening or extended knockdown studies.

Key Elements and Design Rationale

• Multiple independent sequences: Both sets include 3 or 4 distinct siRNA duplexes targeting different regions of the rat Paqr3 mRNA, increasing the probability of achieving effective knockdown and providing built-in sequence redundancy for experimental confidence.
• GAPDH positive control: Included in every set to verify transfection efficiency and confirm that the RNAi machinery is functionally active in the cell model used.
• Scrambled negative control (si-NC): A non-targeting scrambled duplex is included to control for non-specific effects of the transfection reagent and dsRNA delivery.
• FAM-labeled negative control: Fluorescently labeled NC allows direct visualization of transfection efficiency by fluorescence microscopy or flow cytometry prior to mRNA or protein readout.
• HPLC purification: Each duplex is HPLC-purified to remove truncated sequences and reagent impurities that could contribute to off-target effects.

Biological Background

The rat Paqr3 gene (Gene ID: 305203) encodes a protein involved in cellular function in Rattus norvegicus. siRNA-mediated knockdown of Paqr3 in rat cell line models is commonly applied to assess the contribution of this gene to cellular processes including cell signaling, metabolism, and gene regulation. The RNA interference (RNAi) pathway exploits the RISC complex: after cellular entry, the sense (passenger) strand is degraded, and the antisense (guide) strand directs RISC-mediated cleavage of complementary Paqr3 mRNA, leading to mRNA degradation and reduced protein expression.

Research Relevance and Current Trends

• Rat cell line models: siRNA knockdown of Paqr3 in rat cell lines enables loss-of-function studies that complement rat in vivo models, providing a faster and cost-effective approach for initial functional characterization. Rat models are widely used in cardiovascular, neuroscience, and metabolic disease research.
• Pathway validation: After identifying candidate genes in rat transcriptomic or proteomic studies, researchers commonly use siRNA knockdown to confirm the functional role of a target like Paqr3 before advancing to stable KO approaches.
• Target prioritization: The availability of multiple non-overlapping siRNA sequences per set supports orthogonal knockdown confirmation, reducing false-positive conclusions in gene function studies.

Common Research Applications

• mRNA knockdown validation by RT-qPCR: Following transfection, mRNA levels of Paqr3 are typically measured at 24–72 h post-transfection using RT-qPCR, with knockdown efficiency calculated relative to a rat reference gene (e.g., Actb or Gapdh).
• Protein-level knockdown by Western blot: Knockdown at the protein level is assessed 48–96 h post-transfection. Note that mRNA and protein knockdown kinetics may differ depending on protein turnover rate.
• Phenotypic assays: Once knockdown is confirmed, researchers use the optimized siRNA sequence to assess effects on cell viability, proliferation, migration, apoptosis, or signaling pathway activation in rat cell models.
• FAM-NC-guided optimization: The FAM-labeled negative control enables parallel assessment of transfection efficiency (≥80% is typically required) before committing target-specific siRNA to experiments.

Notes for Experimental Interpretation

• Species specificity: These siRNA sequences are designed for rat Paqr3 mRNA. They are not expected to achieve equivalent knockdown in human or mouse cell lines due to coding sequence divergence between species.
• Transfection efficiency: The FAM-labeled NC should be used first to optimize reagent and siRNA concentrations for the specific rat cell line used.
• Off-target effects: Inclusion of the scrambled negative control and comparison of multiple independent Paqr3 siRNA sequences helps distinguish on-target from off-target phenotypes.
• mRNA vs. protein knockdown: Both mRNA (RT-qPCR) and protein (WB) readouts are recommended for complete characterization.

Kit Components

Set A (SI210050A) — 3 siRNA sequences × 5 nmol

• Rat Paqr3 siRNA-1: 5 nmol (HPLC)
• Rat Paqr3 siRNA-2: 5 nmol (HPLC)
• Rat Paqr3 siRNA-3: 5 nmol (HPLC)
• GAPDH siRNA Positive Control: 2.5 nmol (HPLC)
• siRNA Negative Control: 2.5 nmol (HPLC)
• FAM-labeled siRNA Negative Control: 2.5 nmol (HPLC)

Set B (SI210050B) — 4 siRNA sequences × 10 nmol

• Rat Paqr3 siRNA-1: 10 nmol (HPLC)
• Rat Paqr3 siRNA-2: 10 nmol (HPLC)
• Rat Paqr3 siRNA-3: 10 nmol (HPLC)
• Rat Paqr3 siRNA-4: 10 nmol (HPLC)
• GAPDH siRNA Positive Control: 2.5 nmol (HPLC)
• siRNA Negative Control: 2.5 nmol (HPLC)
• FAM-labeled siRNA Negative Control: 2.5 nmol (HPLC)

• Instructions for Pre-designed siRNA Set (PDF)

siRNA Mechanism of Action

Small interfering RNA (siRNA) is a class of chemically synthesized, double-stranded RNA molecule typically 21–23 nucleotides in length. Each duplex consists of two strands:

• Passenger strand (sense strand) — matches the target mRNA sequence; degraded after cellular entry.
• Guide strand (antisense strand) — complementary to the target mRNA; directs the silencing machinery.

Intracellular Pathway

1. Delivery: siRNA duplexes are introduced into cells via a transfection reagent or other delivery method, enabling endosomal escape into the cytoplasm.
2. RISC loading: The duplex is incorporated into the RNA-induced silencing complex (RISC). The passenger strand is cleaved and discarded; the guide strand is retained.
3. Target recognition: The guide strand base-pairs with complementary sequences in the target mRNA through Watson–Crick hybridization.
4. mRNA cleavage: The Argonaute 2 (AGO2) endonuclease within RISC cleaves the target mRNA at the site of complementarity, triggering its degradation.
5. Protein reduction: Loss of target mRNA reduces ribosomal translation, leading to decreased protein expression — typically detectable 48–96 hours post-transfection.

Key Design Features of This Set

• Rat-specific sequences: 3 (Set A) or 4 (Set B) independent siRNA duplexes designed against distinct regions of the rat target mRNA. These sequences are optimized for Rattus norvegicus and are not intended for use in human or mouse cell lines.
• HPLC purification: Chromatographic purification removes truncated oligonucleotides and synthesis by-products that could trigger non-specific immune responses or off-target effects.
• Lyophilized format: Supplied as dry powder for stable ambient-temperature shipping; reconstituted in DEPC (RNase-free) water prior to use.

Overview

The protocol below uses a 24-well plate as the reference format. Scale reagent volumes proportionally for other plate formats using the table provided. All amounts are calculated per well.

Before You Begin

• Reconstitute lyophilized siRNA in DEPC (RNase-free) water to a 20 µM stock concentration (1 OD ≈ 2.5 nmol; add 150 µL DEPC water per 1 OD to reach 20 µM).
• Centrifuge each tube at 10,000 rpm for 30 seconds before opening to concentrate powder at the bottom.
• Prepare single-use aliquots and store at −20°C to avoid repeated freeze–thaw cycles.
• Seed adherent rat cells at 0.5–2.0 × 10⁵ cells/well in 500 µL complete medium one day before transfection. Optimal cell density at transfection: 60–80% confluence.

Transfection Procedure (24-well format)

1. Place transfection reagent at room temperature; mix gently before use.
2. Prepare Tube A (reagent mix): Add 2 µL transfection reagent to 50 µL serum-free medium or Opti-MEM. Mix gently; incubate 5 min at room temperature.
3. Prepare Tube B (siRNA mix): Add 2 µL siRNA stock (20 µM = 40 pmol) to 50 µL serum-free medium or Opti-MEM. Mix gently; incubate 5 min at room temperature.
4. Combine: Add Tube A to Tube B, mix gently, and incubate 15–20 min at room temperature to allow complex formation.
5. Add 100 µL of the transfection complex to each well. Shake plate gently to distribute evenly.
6. After 6–8 hours, replace medium with complete growth medium.
7. Incubate cells and harvest at the appropriate time point for your readout.

Recommended Harvest Time Points

• mRNA knockdown (RT-qPCR): 24–72 hours post-transfection
• Protein knockdown (Western blot / ELISA): 48–96 hours post-transfection

Scale Reference — Reagent Amounts by Plate Format

Note: 20 µM is the recommended storage concentration; 2 µL of 20 µM stock contains 40 pmol siRNA.

Step 1 — Confirm Transfection Efficiency (FAM-NC)

Before evaluating Paqr3 knockdown, confirm that transfection efficiency is ≥80% using the included FAM-labeled negative control. Transfect rat cells with FAM-NC under the same conditions as your target siRNA, then compare bright-field and dark-field (fluorescence) images 6–8 hours post-transfection. If ≥80% of cells are fluorescent under normal growth conditions, transfection efficiency is sufficient to proceed.

Low transfection efficiency is the most common cause of insufficient knockdown — always verify this step before interpreting Paqr3 silencing results.

Step 2 — Assess mRNA Knockdown by RT-qPCR

Harvest cells 24–72 hours post-transfection and extract total RNA. Run RT-qPCR for Paqr3 using a rat reference gene (e.g., Actb or Gapdh) for normalization. Calculate knockdown efficiency using the ΔΔCt method:

• ΔCt = Ct_{target (Paqr3)} − Ct_{reference gene}
• ΔΔCt = ΔCt_{siRNA group} − ΔCt_{negative control group}
• Knockdown efficiency = (1 − 2^−ΔΔCt) × 100%

Evaluate results from all siRNA sequences in the set independently. A knockdown efficiency of ≥70% in at least one sequence confirms effective silencing of Paqr3.

Step 3 — Assess Protein Knockdown by Western Blot

Harvest cells 48–96 hours post-transfection for protein-level analysis. Run Western blot using an appropriate anti-Paqr3 antibody that detects the rat protein, with Gapdh or another housekeeping protein as a loading control. Note that mRNA and protein knockdown kinetics may differ depending on the half-life of the Paqr3 protein.

Interpreting Your Controls

Knockdown Performance Guarantee

When transfection efficiency reaches ≥80% (confirmed by FAM-labeled negative control), we guarantee that at least one of the siRNA sequences in the set will achieve ≥70% knockdown efficiency at the mRNA level.

What Happens If the Guarantee Is Not Met

1. Free redesign (first round): If the guaranteed knockdown is not achieved, we will redesign and resynthesize 2 new siRNA sequences at no charge.
2. Refund (if redesign also fails): If the resynthesized sequences still do not achieve the guaranteed knockdown, a full refund will be issued.

Requirements to Claim the Guarantee

• FAM-NC transfection images: Bright-field and dark-field (fluorescence) images confirming ≥80% transfection efficiency in the rat cell model used.
• RT-qPCR raw data: Raw Ct values for the target gene and reference gene across all siRNA groups, negative control, and mock group, demonstrating that knockdown did not meet the ≥70% threshold.

Important Limitations

• The guarantee applies to mRNA-level knockdown only. Protein-level results (e.g., Western blot alone) are not sufficient to claim the guarantee.
• If only Western blot data is provided, one free redesign will be offered; however, refunds do not apply based on protein data alone.
• The guarantee assumes standard transfection conditions as described in the transfection protocol.

Contact Us

For guarantee claims or return inquiries, please contact us at support@biohippo.com. Please include your order number, rat cell line used, and all required documentation when submitting a claim.

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