TNRC6C antibodies are polyclonal or monoclonal antibodies developed to target the TNRC6C protein, which facilitates miRNA-mediated gene silencing by bridging Argonaute proteins and deadenylase complexes . These antibodies are widely used in immunohistochemistry (IHC), Western blotting, and immunofluorescence to study TNRC6C expression patterns in cancer biology, particularly in thyroid malignancies.
TNRC6C antibodies have enabled critical discoveries through the following methodologies:
Immunohistochemistry (IHC): Detects TNRC6C protein localization and expression levels in PTC tissues versus adjacent normal tissues .
Western Blotting: Validates TNRC6C knockdown or overexpression in cell lines like BCPAP and TPC1 .
Functional Assays: Correlates TNRC6C expression with cellular behaviors (proliferation, apoptosis, migration) in vitro .
TNRC6C is downregulated in PTC tissues compared to normal thyroid tissues (IHC validation) .
Overexpression of TNRC6C inhibits PTC cell proliferation by 30–40%, reduces migration/invasion by 50–60%, and increases apoptosis by 6-fold .
Low TNRC6C correlates with larger tumors, lymph node metastasis, and advanced clinical stages (Table 1) .
RNA sequencing and TCGA data identified 12 oncogenic genes (e.g., CTHRC1, MMP14, COL1A1) negatively regulated by TNRC6C. These genes are overexpressed in PTC and linked to aggressive phenotypes .
Gene | Function in Cancer | Regulation by TNRC6C |
---|---|---|
CTHRC1 | Promotes metastasis | Downregulated |
MMP14 | Enhances extracellular remodeling | Downregulated |
COL1A1 | Facilitates tumor stroma formation | Downregulated |
Prognostic Marker: Low TNRC6C expression predicts poor outcomes in PTC patients .
Therapeutic Target: Restoring TNRC6C expression suppresses tumor growth in vitro, suggesting potential for gene therapy .
Antisense RNA Interaction: TNRC6C-AS1, a lncRNA, inversely regulates TNRC6C and could be targeted to modulate its activity .
TNRC6C (Trinucleotide Repeat Containing 6C) functions as an important component within miRNA-induced silencing complex . It has been identified as a potential tumor suppressor in papillary thyroid cancer (PTC), with studies showing that it inhibits cell proliferation, migration, and invasion while promoting apoptosis in PTC cells . The protein's role in post-transcriptional gene regulation makes it a valuable target for cancer research, particularly in understanding gene expression control mechanisms that contribute to tumorigenesis.
TNRC6C antibodies are validated for multiple experimental applications including ELISA, Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) . Different commercially available antibodies show varying application profiles - for example, Sigma-Aldrich's HPA062051 antibody is specifically validated for immunofluorescence at concentrations of 0.25-2 μg/mL . When selecting an antibody, researchers should verify validation data for their specific application to ensure reliable results.
TNRC6C antibodies are typically shipped on wet ice and should be stored at -20°C for optimal preservation . Most commercial TNRC6C antibodies are provided in buffered aqueous glycerol solutions to maintain stability . Researchers should avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt. For short-term use (1-2 weeks), storage at 4°C is acceptable, but long-term storage requires -20°C conditions to prevent degradation and maintain binding efficacy across multiple experiments.
When validating TNRC6C antibodies, researchers should use:
Positive Controls:
PTC cell lines with known TNRC6C expression such as BCPAP or TPC1 cells
HEK293 cells transfected with TNRC6C expression vectors
Negative Controls:
TNRC6C siRNA-transfected cells showing knockdown (validation by qPCR)
Secondary antibody-only controls to assess non-specific binding
Isotype controls with irrelevant antibodies of the same class/species
Validation should include multiple controls to ensure specificity before undertaking extensive experimental work.
Optimizing IHC for TNRC6C in thyroid tissues requires attention to several parameters:
Recommended Protocol:
Fixation: Use 10% neutral buffered formalin for 24-48 hours
Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes
Blocking: 5% normal goat serum in PBS for 1 hour at room temperature
Primary antibody incubation: Anti-TNRC6C at 1-2 μg/mL overnight at 4°C
Detection system: HRP-polymer based with DAB chromogen
Counterstaining: Hematoxylin for 30 seconds
Critical Considerations:
Nuclear localization of TNRC6C requires careful optimization of permeabilization steps
Comparison between tumor and adjacent normal tissue on the same slide helps control for technical variations
Quantification should include both intensity and percentage of positive cells using standardized scoring systems
Investigating TNRC6C's role in miRNA-induced silencing requires multifaceted approaches:
Recommended Methodologies:
Co-Immunoprecipitation (Co-IP):
Use anti-TNRC6C antibody to pull down the protein complex
Analyze interacting partners by mass spectrometry or Western blot
Confirm interactions with Argonaute proteins
RNA-Immunoprecipitation (RIP):
Immunoprecipitate TNRC6C-containing complexes
Extract and identify associated miRNAs and mRNAs by sequencing
Microscopy techniques:
Immunofluorescence co-localization with other miRISC components
Live-cell imaging with fluorescently tagged TNRC6C
Functional assays:
Distinguishing between TNRC6 family members requires careful antibody selection and validation:
Recommended Approaches:
Antibody epitope analysis:
Paralog-specific knockdown controls:
siRNA targeting each paralog independently
Western blot with different antibodies to confirm specificity
Recombinant protein standards:
Include purified TNRC6A/B/C proteins as controls
Assess cross-reactivity quantitatively
Multiple lines of evidence establish TNRC6C's tumor suppressor role:
Experimental Evidence:
Expression analysis: TNRC6C is downregulated in PTC compared to normal thyroid tissue
Functional studies:
Clinical correlations:
Mechanistic insights:
These findings collectively demonstrate that TNRC6C functions to suppress thyroid cancer progression and may serve as a therapeutic target and prognostic marker for PTC patients.
TNRC6C-AS1 is a long non-coding RNA that is distinct from the TNRC6C protein:
Key Differences and Research Implications:
Molecular nature:
Expression patterns:
Functional roles:
Experimental considerations:
Antibody-based detection only works for TNRC6C protein, not the AS1 transcript
RNA-based methods (qRT-PCR) are needed to study TNRC6C-AS1
Researchers must carefully distinguish between these entities in experimental design
Mechanistic interaction:
Researchers encountering contradictory findings should implement these methodological approaches:
Reconciliation Strategies:
Comprehensive tissue analysis:
Compare matched tumor-normal pairs from the same patients
Analyze different histological subtypes separately
Use laser capture microdissection to isolate specific cell populations
Multi-level expression analysis:
Technical standardization:
Use consistent antibody clones and detection protocols
Implement quantitative scoring systems with defined thresholds
Include multiple positive and negative controls
Integrative data analysis:
Compare findings with public datasets (TCGA, GEO)
Perform meta-analysis of published studies
Correlate with clinical parameters to identify context-dependent effects
Researchers frequently encounter these challenges with TNRC6C Western blotting:
Common Issues and Solutions:
Issue | Possible Causes | Solution |
---|---|---|
High molecular weight band detection difficulties | Large protein size (175 kDa) | Use gradient gels (4-12%); Extend transfer time (overnight at low voltage) |
Multiple bands/non-specific binding | Cross-reactivity with TNRC6A/B | Use validated antibodies with confirmed specificity; Include knockout controls |
Weak signal | Low expression levels | Increase protein loading (50-80 μg); Use enhanced chemiluminescence detection systems |
Inconsistent results between experiments | Protein degradation | Add additional protease inhibitors; Minimize freeze-thaw cycles of samples |
Background issues | Secondary antibody cross-reactivity | Optimize blocking (5% BSA often better than milk for phospho-antibodies); Increase washing steps |
Optimized Western Blot Protocol for TNRC6C:
Use fresh lysates with complete protease inhibitor cocktails
Load 60-80 μg protein per lane on 4-8% gradient gels
Transfer at 30V overnight at 4°C to PVDF membranes
Block with 5% BSA for 2 hours at room temperature
Incubate with anti-TNRC6C antibody (1:1000) overnight at 4°C
Wash extensively (4 × 10 min) with TBST
Use HRP-conjugated secondary antibody (1:5000) for 1 hour
Develop using enhanced chemiluminescence reagents
Precise subcellular localization of TNRC6C requires these optimization steps:
Optimized Immunofluorescence Protocol:
Cell preparation:
Culture cells on poly-L-lysine coated coverslips
Fix with 4% paraformaldehyde (10 minutes at room temperature)
Permeabilize with 0.2% Triton X-100 (for nuclear proteins)
Antibody incubation:
Nuclear co-staining:
Include DAPI (1 μg/mL) for nuclear visualization
Consider co-staining with other miRISC components
Image acquisition:
Use confocal microscopy for precise localization
Z-stacking to confirm true nuclear vs. cytoplasmic distribution
Consistent exposure settings between samples
Controls:
Include TNRC6C knockdown cells
Secondary-only controls
Known subcellular markers co-staining
The IHC results showed that TNRC6C is primarily localized in the nucleus, suggesting it may regulate gene expression at the transcriptional level in addition to its known role in miRNA-induced silencing complexes .