DICER1 Antibody

What is DICER1 and why is it important in biomedical research?

DICER1, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme encoded by the DICER1 gene located on chromosome 14q32.13 in humans. It belongs to the ribonuclease III (RNaseIII) family and plays a crucial role in microRNA (miRNA) biogenesis and small RNA processing pathways .

DICER1 functions as a key component of the RNA-induced silencing complex (RISC) loading complex (RLC), working alongside EIF2C2/AGO2 and TARBP2 to process precursor miRNAs (pre-miRNAs) into mature miRNAs. Within this complex, DICER1 cleaves double-stranded RNA to produce short interfering RNAs (siRNAs) that target the selective destruction of complementary RNAs .

The significance of DICER1 in research extends beyond basic RNA processing mechanisms to its role in:

• Cancer biology: DICER1 functions as a tumor suppressor, with mutations associated with DICER1 syndrome, a rare genetic disorder predisposing to multiple tumor types

• Immune regulation: DICER1 expression levels influence immune-related gene networks

• Development and metabolism: Phosphorylation of DICER1 has been linked to accelerated metabolism, aging, and fertility issues

What are the essential characteristics of DICER1 antibodies researchers should know?

DICER1 antibodies possess several key characteristics that researchers should consider when designing experiments:

When selecting a DICER1 antibody, researchers should be aware that:

• The detection of both 220-250 kDa and 90 kDa bands is common and may represent different isoforms or processing products

• Phosphorylated DICER1 exhibits predominantly nuclear localization, while total DICER1 shows more variable distribution

• Different epitope targets may yield varying staining patterns, particularly in fixed tissues where epitope accessibility can be affected

How should DICER1 antibody dilutions be optimized for different experimental applications?

Optimizing DICER1 antibody dilutions is critical for obtaining specific signals with minimal background. Based on validated protocols, here are recommended dilution ranges for different applications:

Methodological considerations for optimization:

• Always perform a dilution series to determine optimal concentration for your specific sample type

• Include positive controls (e.g., HepG2, K-562, or HeLa cells for WB applications)

• For IHC applications, boiling paraffin sections in 10mM citrate buffer (pH 6.0) for 20 minutes is often required for optimal staining

• For phospho-specific DICER1 detection, include phosphatase inhibitors during sample preparation

• When using fluorescently-conjugated antibodies (e.g., CoraLite® Plus 488), protect from light exposure and use appropriate emission/excitation settings (493nm/522nm)

How can DICER1 antibodies be used to investigate DICER1 syndrome and associated tumors?

DICER1 syndrome is a rare genetic disorder characterized by pathogenic germline variants in the DICER1 gene that predispose individuals to a wide spectrum of tumors. DICER1 antibodies provide valuable tools for investigating this syndrome through multiple approaches:

DICER1 syndrome characteristics relevant to antibody-based studies:

• Associated with pleuropulmonary blastoma (PPB), thyroid cancer, ovarian sex-cord stromal tumors, and cystic nephroma

• Majority of tumors occur in infancy, childhood, and adolescence

• Follows a "two-hit" mechanism where germline DICER1 mutations are accompanied by somatic "second hits"

Methodological approaches using DICER1 antibodies:

• Tumor Characterization:

  • IHC analysis of tumor specimens to assess DICER1 expression patterns

  • Compare expression between normal and neoplastic tissues

  • Correlate with histopathological features and clinical outcomes

• Surveillance and Early Detection:

  • Pulmonary imaging surveillance is supported by significant differences in survival between type I PPB (79% 5-year survival) versus type II (59%) and type III (37%)

  • DICER1 antibodies can help characterize early neoplastic changes in resected suspicious lesions

• Research considerations for DICER1 syndrome studies:

  • Approximately 95% of non-index case individuals with germline pathogenic DICER1 variants do not develop tumors by age 10

  • The incidence of loss-of-function (LOF) variants in population datasets is approximately 1:5121

  • Consider both the core and extended surveillance programs when designing research protocols

• Emerging research directions:

  • Investigating genotype-phenotype correlations using antibodies to assess how specific DICER1 mutations affect protein expression and localization

  • Exploring novel biomarkers that could complement imaging surveillance

What role does DICER1 phosphorylation play in disease processes and how can this be studied?

DICER1 phosphorylation represents a critical post-translational modification with significant implications for cellular function and disease pathogenesis. Research has demonstrated that DICER1 is phosphorylated by the ERK-MAP kinase pathway, with important functional consequences:

Key findings on DICER1 phosphorylation:

• Phosphorylation occurs at two conserved serine residues (S1712 and S1836 in mice)

• Constitutive phosphorylation accelerates metabolism and is associated with aging, infertility, and metabolic disorders

• In human endometrioid cancers, phosphorylated DICER1 is significantly associated with invasive disease

Methodological approaches for studying phosphorylated DICER1:

• Phospho-specific antibody applications:

  • Use phospho-DICER1 specific antibodies for detecting the phosphorylated protein

  • Compare with total DICER1 antibodies to determine phosphorylation ratios

  • Immunofluorescence studies reveal that phosphorylated DICER1 appears predominantly nuclear in localization

• Clinical correlation studies:

  • Tissue microarray analysis using phospho-DICER1 antibodies can reveal clinical associations

  • Published data shows significant correlations between nuclear phospho-DICER1 positivity and:

• Experimental models:

  • Use of phospho-mimetic mutations (S1712D and S1836D) to study constitutive phosphorylation effects

  • These models demonstrate that phospho-mimetic Dicer1 promotes tumor development and invasion in multiple cancer models

  • Quantification of cells with nuclear Dicer1 in tissue sections can be performed manually, with cells showing >50% nuclear Dicer1 staining considered positive

How do DICER1 antibodies contribute to understanding the role of microRNA processing in disease?

DICER1's central role in microRNA (miRNA) processing makes it a key target for investigating dysregulated RNA metabolism in various diseases. DICER1 antibodies provide valuable tools for examining these processes:

DICER1's role in miRNA processing:

• Processes precursor miRNAs (pre-miRNAs) to mature miRNAs within the RISC loading complex

• Works in conjunction with EIF2C2/AGO2 and TARBP2 to generate functional miRNAs

• Disruptions in this pathway can lead to global or selective miRNA dysregulation

Research applications using DICER1 antibodies:

• DICER1 overexpression studies:

  • Transfection of cells with DICER1 expression vectors allows investigation of dose-dependent effects

  • Recent research demonstrated that DICER1 overexpression in mesenchymal stem cells significantly altered immune-related gene expression profiles

  • Specifically, DICER1 overexpression led to:

    • Increased expression of COX-2, DDX-58, IFIH1, MYD88, RNase L, TLR3/4, and TDO2 (3-16 fold increases)

    • Decreased expression of TSG-6

    • Higher expression levels of multiple interleukins (IL-1, 6, 8, 17, 18), CCL2, INF-γ, TGF-β, and TNF-α

• Protein-RNA interaction studies:

  • IP with DICER1 antibodies can isolate DICER1-associated RNA complexes

  • RNA immunoprecipitation (RIP) protocols using DICER1 antibodies help identify miRNAs being processed

  • Comparing disease models with controls can reveal alterations in DICER1-associated RNA populations

• Subcellular localization studies:

  • IF/ICC applications reveal compartmentalization of DICER1 and its processing activities

  • Changes in localization patterns may indicate altered function in disease states

  • Dilutions of 1:50-1:500 for IF-P and 1:200-1:800 for IF/ICC are recommended

What controls are essential when using DICER1 antibodies in experimental protocols?

Implementing appropriate controls is critical for ensuring reliable results when working with DICER1 antibodies. The following controls should be considered for different experimental applications:

• Positive controls:

  • Cell lines with confirmed DICER1 expression (HepG2, K-562, HeLa cells)

  • Tissues with known DICER1 expression patterns (human testis, lung cancer tissue, ovarian tumor tissue)

  • Process these controls identically to experimental samples

• Negative controls:

  • DICER1 knockdown or knockout samples when available

  • No primary antibody controls for IHC/IF applications

  • Isotype controls (rabbit IgG) at matched concentrations

  • Peptide competition controls (pre-incubation with immunizing peptide)

• Application-specific controls:

• Methodological validation approaches:

  • Cross-validation using multiple antibodies targeting different DICER1 epitopes

  • Correlation with mRNA expression data from RT-PCR or RNA-seq

  • For phospho-specific antibodies, comparison with phosphatase-treated samples

How should researchers interpret multiple bands or unexpected molecular weights in DICER1 Western blots?

Western blot analysis of DICER1 frequently reveals multiple bands or unexpected molecular weights, which requires careful interpretation:

Expected DICER1 band patterns:

• Primary band: 220-250 kDa (full-length protein)

• Secondary band: ~90 kDa (potential processing product or alternative isoform)

• These patterns are consistently reported across multiple antibody sources

Interpretive framework for unusual band patterns:

• Potential explanations for multiple bands:

  • Alternative splicing or protein isoforms

  • Post-translational modifications (phosphorylation, ubiquitination)

  • Proteolytic processing during sample preparation

  • Cross-reactivity with related proteins

• Troubleshooting approach:

  • Validate with multiple antibodies targeting different epitopes

  • Include appropriate controls (DICER1 knockdown/knockout samples)

  • Optimize sample preparation (add protease inhibitors, maintain cold temperatures)

  • Perform peptide competition assays to confirm specificity

• Technical considerations for large proteins like DICER1:

  • Use lower percentage gels (6-8%) for better resolution of high molecular weight bands

  • Extend transfer times for complete transfer of large proteins

  • Consider gradient gels to simultaneously resolve both high and low molecular weight bands

• Interpreting phosphorylation status:

  • Phosphorylated DICER1 may exhibit a slight upward shift in molecular weight

  • When using total DICER1 antibodies, compare with phospho-specific antibody signals on parallel blots

  • Altered band ratios may indicate changes in DICER1 processing or modification

What factors affect DICER1 subcellular localization and how should immunofluorescence results be analyzed?

DICER1 subcellular localization provides important insights into its function and regulation. Several factors influence localization patterns and their interpretation:

Factors affecting DICER1 localization:

• Phosphorylation status: Phosphorylated DICER1 shows predominantly nuclear localization

• Cell type and physiological state: Distribution patterns may vary between tissues and conditions

• Disease state: Altered localization is observed in pathological contexts, particularly in cancers

Methodological considerations for accurate analysis:

• Quantification approach:

  • For nuclear localization studies, cells with >50% of nucleus showing DICER1 staining can be considered positive

  • Multiple fields should be analyzed (e.g., three 20× images from different regions)

  • Conduct blinded assessment to avoid bias

• Technical factors influencing apparent localization:

  • Fixation method: Different fixatives affect epitope accessibility

  • Permeabilization: Insufficient permeabilization may limit antibody access to nuclear DICER1

  • Antibody concentration: Optimal dilutions for IF/ICC range from 1:50-1:800

• Co-localization analysis:

  • Use appropriate nuclear counterstains (e.g., DAPI)

  • Consider co-staining with markers of specific subcellular compartments

  • Phospho-DICER1 shows significant nuclear co-localization, while total DICER1 often displays more diffuse patterns

• Research example from phospho-DICER1 studies:

  • In endometrioid cancer studies, tumors were classified as positive when >10% of cells showed phospho-DICER1 signal

  • This classification revealed significant associations with clinicopathologic factors including BMI, lymphovascular space invasion, and depth of invasion

  • Similar quantitative approaches can be adapted for other DICER1 research contexts

How are DICER1 antibodies being applied to develop new disease surveillance and intervention strategies?

DICER1 antibodies are increasingly valuable for developing novel approaches to disease surveillance and intervention, particularly in DICER1 syndrome-associated conditions:

Current surveillance recommendations for DICER1 pathogenic variant carriers:

• Core surveillance program covers the most common DICER1-associated tumors (~90-95% of reported cases)

• Extended program includes additional procedures for personalized surveillance

• Pulmonary surveillance is particularly important due to significant survival differences between early and advanced pleuropulmonary blastoma

Emerging research applications:

• Biomarker development:

  • IHC analysis of tissue samples to identify early neoplastic changes

  • Correlation of DICER1 expression/modification patterns with disease progression

  • Development of minimally invasive detection methods based on DICER1 alterations

• Therapeutic target identification:

  • Phosphorylated DICER1 represents a potential therapeutic target, particularly in invasive cancers

  • Inhibition of pathways regulating DICER1 phosphorylation (e.g., ERK-MAP kinase pathway)

  • Personalized approaches based on specific DICER1 mutation profiles

• Risk stratification:

  • Using DICER1 antibodies to identify patterns of expression or modification associated with higher risk of tumor development

  • Integration with genetic testing data for comprehensive risk assessment

  • Development of predictive models incorporating molecular and clinicopathological variables

• Therapeutic response monitoring:

  • Assessing changes in DICER1 expression or modification patterns during treatment

  • Potential use as pharmacodynamic biomarkers in clinical trials

  • Correlation with disease progression or regression

What are the latest methodological advances in DICER1 antibody-based research?

Recent methodological advances have expanded the utility and precision of DICER1 antibody-based research:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Live cell imaging using fluorescently-tagged antibody fragments

  • Quantitative image analysis platforms for standardized assessment

  • Multiplex immunofluorescence for simultaneous detection of DICER1 and interacting partners

• Single-cell applications:

  • Integration of DICER1 antibody detection with single-cell sequencing

  • Flow cytometry protocols for intracellular DICER1 detection in rare cell populations

  • Correlation of DICER1 protein levels with transcriptomic profiles at single-cell resolution

• Engineered antibody approaches:

  • Development of highly specific monoclonal antibodies targeting particular DICER1 epitopes

  • Phospho-specific antibodies detecting distinct phosphorylation sites (S1712, S1836)

  • Fluorescently conjugated antibodies (e.g., CoraLite® Plus 488) for direct detection

• Specialized applications:

  • Chromatin immunoprecipitation (ChIP) protocols for investigating DICER1 interactions with chromatin

  • Proximity ligation assays for detecting DICER1 protein-protein interactions in situ

  • Mass spectrometry-based approaches for comprehensive analysis of DICER1 modifications