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Overview
This pre-designed siRNA set enables sequence-specific knockdown of the human MCFD2 gene (NCBI Gene ID: 90411) 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 (SI009345A, 3 siRNA sequences at 3×5 nmol) and Set B (SI009345B, 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 MCFD2 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 MCFD2 gene (Gene ID: 90411) encodes a human protein involved in cellular function. siRNA-mediated knockdown of MCFD2 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 MCFD2 mRNA, leading to mRNA degradation and reduced protein expression.
Research Relevance and Current Trends
- Functional genomics screens: Pre-designed siRNA sets targeting individual genes such as MCFD2 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 MCFD2 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 MCFD2 are typically measured at 24–72 h post-transfection using RT-qPCR, with knockdown efficiency calculated relative to a reference gene.
- 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.
- 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
- 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: Inclusion of the scrambled negative control and comparison of multiple independent MCFD2 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.
- Cell-type considerations: Knockdown efficiency may vary across different human cell lines due to differences in transfection efficiency, RNAi machinery activity, and baseline MCFD2 expression levels.
Kit Components
Set A (SI009345A) — 3 siRNA sequences × 5 nmol
- Human MCFD2 siRNA-1: 5 nmol (HPLC)
- Human MCFD2 siRNA-2: 5 nmol (HPLC)
- Human MCFD2 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 (SI009345B) — 4 siRNA sequences × 10 nmol
- Human MCFD2 siRNA-1: 10 nmol (HPLC)
- Human MCFD2 siRNA-2: 10 nmol (HPLC)
- Human MCFD2 siRNA-3: 10 nmol (HPLC)
- Human MCFD2 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)
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
- Delivery: siRNA duplexes are introduced into cells via a transfection reagent or other delivery method, enabling endosomal escape into the cytoplasm.
- 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.
- Target recognition: The guide strand base-pairs with complementary sequences in the target mRNA through Watson–Crick hybridization.
- mRNA cleavage: The Argonaute 2 (AGO2) endonuclease within RISC cleaves the target mRNA at the site of complementarity, triggering its degradation.
- 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)
- Place transfection reagent at room temperature; mix gently before use.
- 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.
- 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.
- Combine: Add Tube A to Tube B, mix gently, and incubate 15–20 min at room temperature to allow complex formation.
- Add 100 µL of the transfection complex to each well. Shake plate gently to distribute evenly.
- After 6–8 hours, replace medium with complete growth medium.
- 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
| Plate Format | Well Area | Medium Volume | Opti-MEM (×2 tubes) | siRNA Amount | Transfection Reagent |
|---|---|---|---|---|---|
| 96-well | 0.3 cm² | 100 µL | 2 × 10 µL | 20 pmol | 1 µL |
| 24-well | 2.0 cm² | 500 µL | 2 × 50 µL | 40 pmol | 2 µL |
| 12-well | 4.0 cm² | 1 mL | 2 × 100 µL | 80 pmol | 4 µL |
| 6-well | 10.0 cm² | 2 mL | 2 × 200 µL | 150 pmol | 7.5 µL |
| 60 mm dish | 20.0 cm² | 5 mL | 2 × 500 µL | 300 pmol | 15 µL |
| 10 cm dish | 60.0 cm² | 15 mL | 2 × 1 mL | 600 pmol | 30 µL |
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 MCFD2 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 MCFD2 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 MCFD2 using a reference gene (e.g., beta-actin or GAPDH) for normalization. Calculate knockdown efficiency using the ΔΔCt method:
- ΔCt = Cttarget (MCFD2) − Ctreference gene
- ΔΔCt = ΔCtsiRNA group − ΔCtnegative 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 MCFD2.
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-MCFD2 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 MCFD2 protein.
Interpreting Your Controls
| Control | Expected Result | If Result Differs |
|---|---|---|
| Negative control (si-NC) | No knockdown of MCFD2; <20% difference vs. mock group | Investigate non-specific effects of transfection reagent or dsRNA |
| GAPDH positive control | ≥70% knockdown of GAPDH mRNA | RNAi pathway may be impaired; check transfection conditions |
| Mock group (no siRNA) | Within ±20% of si-NC group | If outside range, investigate reagent toxicity or handling variables |
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
- Free redesign (first round): If the guaranteed knockdown is not achieved, we will redesign and resynthesize 2 new siRNA sequences at no charge.
- 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.
Need a configuration beyond the standard pre-designed siRNA set? We can help tailor your order for screening, validation, or scale-up studies. Custom and add-on options may include your preferred set format (Set A with 3 target-gene siRNAs at 5 nmol each, or Set B with 4 target-gene siRNAs at 10 nmol each), additional target-specific siRNA tubes, repeat or bulk packaging, and extra control reagents such as GAPDH siRNA positive control, negative control, or FAM-labeled negative control. We can also discuss larger synthesis scales, project-based quantity planning, and supply preferences aligned with the standard product format, including HPLC-purified, lyophilized siRNA for DEPC water reconstitution. For custom requests or add-on inquiries, please contact us at support@biohippo.com. Our team will review your study needs and get back to you with the best-fit option.
Methodology References
- Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–811. https://doi.org/10.1038/35888
- Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411(6836):494–498. https://doi.org/10.1038/35078107
- Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. Nature Biotechnology. 2004;22(3):326–330. https://doi.org/10.1038/nbt936
- Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nature Reviews Drug Discovery. 2010;9(1):57–67. https://doi.org/10.1038/nrd3054
- Kaelin WG Jr. Use and abuse of RNAi to study mammalian gene function. Science. 2012;337(6093):421–422. https://doi.org/10.1126/science.1225150
Gene-Specific Resources — MCFD2
- NCBI Gene: https://www.ncbi.nlm.nih.gov/gene/90411 — Official gene record, reference sequences, isoforms, and expression data for human MCFD2.
- PubMed — siRNA knockdown literature: Search PubMed for MCFD2 siRNA studies — Browse publications on MCFD2 knockdown, functional studies, and pathway roles.
- UniProtKB: Search UniProt for human MCFD2 protein — Protein function, domains, post-translational modifications, and interaction data.
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