Human DHFR Pre-designed siRNA

Overview

This pre-designed siRNA set enables sequence-specific knockdown of the human DHFR gene (NCBI Gene ID: 1719) in human 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 (SI004247A, 3 siRNA sequences at 3×5 nmol) and Set B (SI004247B, 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 DHFR 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 DHFR gene (Gene ID: 1719) encodes a human protein involved in cellular function. siRNA-mediated knockdown of DHFR is commonly applied to assess the contribution of this gene to cell proliferation, signaling, or metabolic pathways depending on the biological context. 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 DHFR mRNA, leading to mRNA degradation and reduced protein expression. This approach is widely used to model loss-of-function phenotypes in cultured human cell lines.

Research Relevance and Current Trends

• Functional genomics screens: Pre-designed siRNA sets targeting individual genes such as DHFR are routinely used in arrayed or pooled functional screens to identify gene dependencies in cancer cell lines, primary cells, and disease models.
• Pathway validation: After identifying candidate genes by transcriptomics or proteomics, researchers commonly use siRNA knockdown to confirm the functional role of a target like DHFR before advancing to stable KO models.
• Target prioritization: The availability of multiple non-overlapping siRNA sequences per set supports orthogonal knockdown confirmation, a best practice for reducing false-positive conclusions in gene function studies.

Common Research Applications

• mRNA knockdown validation by RT-qPCR: Following transfection, mRNA levels of DHFR are typically measured at 24–72 h post-transfection using RT-qPCR, with knockdown efficiency calculated relative to a reference gene. Multiple siRNA sequences allow identification of the most effective duplex for downstream studies.
• Protein-level knockdown by Western blot: Knockdown at the protein level is assessed 48–96 h post-transfection by Western blot or ELISA. 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 depending on the role of DHFR in the model studied.
• FAM-NC-guided optimization: The FAM-labeled negative control enables parallel assessment of transfection efficiency (≥80% is typically required for reliable knockdown results) before committing target-specific siRNA to experiments.

Notes for Experimental Interpretation

• Transfection efficiency: Knockdown outcomes are highly dependent on achieving adequate transfection efficiency. The FAM-labeled NC should be used first to optimize reagent and siRNA concentrations for the specific cell line used.
• Off-target effects: Even HPLC-purified siRNAs can exhibit sequence-dependent off-target activity. Inclusion of the scrambled negative control and comparison of multiple independent DHFR siRNA sequences helps distinguish on-target from off-target phenotypes.
• mRNA vs. protein knockdown: High mRNA knockdown does not always correlate with equivalent protein reduction, depending on protein half-life. Both mRNA (RT-qPCR) and protein (WB) readouts are recommended for complete characterization.
• Cell-type considerations: Knockdown efficiency and phenotypic outcomes may vary across different human cell lines due to differences in transfection efficiency, RNAi machinery activity, and baseline DHFR expression levels.

Kit Components

Set A (SI004247A) — 3 siRNA sequences × 5 nmol

• Human DHFR siRNA-1: 5 nmol (HPLC)
• Human DHFR siRNA-2: 5 nmol (HPLC)
• Human DHFR 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 (SI004247B) — 4 siRNA sequences × 10 nmol

• Human DHFR siRNA-1: 10 nmol (HPLC)
• Human DHFR siRNA-2: 10 nmol (HPLC)
• Human DHFR siRNA-3: 10 nmol (HPLC)
• Human DHFR 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

• Multiple sequences: 3 (Set A) or 4 (Set B) independent siRNA duplexes targeting distinct regions of the same mRNA increase the probability of effective silencing and support orthogonal confirmation.
• 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 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 DHFR knockdown, confirm that transfection efficiency is ≥80% using the included FAM-labeled negative control. Transfect 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 DHFR 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 DHFR using a reference gene (e.g., beta-actin or GAPDH) for normalization. Calculate knockdown efficiency using the ΔΔCt method:

• ΔCt = Ct_{target (DHFR)} − 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 DHFR.

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-DHFR antibody, 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 DHFR 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 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, cell line used, and all required documentation when submitting a claim.

Can't find the siRNA you're looking for? We offer custom and add-on services to help you move your project forward, from target sequence design and optimization to custom siRNA duplex synthesis for both in vitro and in vivo studies. Options may include standard or chemically modified siRNA, HPLC purification, full QC documentation, multiple synthesis scales from screening quantities to larger batches, as well as labels, conjugation, and other modifications to support stability, tracking, and delivery. We can also help with pre-designed siRNA sets, control formats, and related RNA solutions when a catalog item is not available. Click Talk to a Scientist to submit a request, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.