RNASEH2B Antibody

What is RNASEH2B and why is it important in research?

RNASEH2B is one of three subunits (along with RNASEH2A and RNASEH2C) that form the RNase H2 complex. This enzyme is crucial for:

• Breaking down RNA:DNA hybrids formed during DNA replication

• Removing misincorporated ribonucleotides from genomic DNA through ribonucleotide excision repair (RER)

• Maintaining genomic stability

• Preventing inappropriate immune activation

The importance of RNASEH2B is highlighted by its involvement in Aicardi-Goutières syndrome when mutated, and its emerging role in cancer biology. Loss of RNASEH2B has been found to sensitize cells to PARP inhibition, suggesting therapeutic implications . Additionally, RNASEH2B is required for efficient LINE-1 retrotransposition, which is relevant to both normal cellular processes and disease states .

What types of RNASEH2B antibodies are available for research?

Based on current research applications, RNASEH2B antibodies available include:

Most validated antibodies have been tested in human, mouse, and rat samples, with observed molecular weight around 35-37 kDa . When selecting an antibody, researchers should consider validation status for their specific application and species of interest.

How should RNASEH2B antibodies be optimized for Western blot analysis?

For optimal Western blot results with RNASEH2B antibodies:

• Sample preparation:

  • Use appropriate lysis buffers (RIPA buffer with protease inhibitors works effectively)

  • Ensure complete denaturation at 95°C for 5 minutes

• Running conditions:

  • Use 10-12% SDS-PAGE gels

  • Look for bands at approximately 35-37 kDa

• Antibody dilution:

  • Start with 1:500-1:1000 dilution and optimize as needed

  • Incubate overnight at 4°C for best results

• Controls:

  • Include positive controls (HeLa, MCF-7 cells, mouse thymus tissue)

  • Use RNASEH2B knockout or siRNA-treated samples as negative controls

  • CRISPR-Cas9 generated RNASEH2A knockout cell lines also show reduced RNASEH2B levels

• Signal detection:

  • Both chemiluminescent and fluorescent secondary antibodies are suitable

  • Longer exposure times may be needed for detecting endogenous levels in some tissues

When troubleshooting, remember that RNASEH2B levels can change with replication stress, so cellular conditions may affect expression levels .

What are the best protocols for RNASEH2B immunohistochemistry (IHC)?

For successful RNASEH2B IHC:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections (4-5 μm thick)

  • Antigen retrieval is critical: use citrate buffer (pH 6.0) with heat-induced epitope retrieval

• Antibody conditions:

  • Validated antibodies show predominantly nuclear staining pattern

  • Automated colorimetric digital (HALO) and visual analyses show good correlation

• Evaluation parameters:

  • Score nuclear H-score for analysis as RNASEH2B is predominantly nuclear

  • Both homogeneous and heterogeneous RNASEH2B protein loss can be observed in clinical samples

• Technical considerations:

  • Bone decalcification protocols (particularly EDTA) may affect RNASEH2B staining

  • Internal controls should be assessed for protein preservation quality

In clinical studies, RNASEH2B IHC has shown 93% sensitivity and 71% specificity for detecting RNASEH2B homozygous deletions in uterine leiomyosarcoma .

How can I validate the specificity of RNASEH2B antibodies?

Multiple validation approaches should be employed:

• siRNA knockdown validation:

  • Transfect cells with RNASEH2B-targeting siRNA

  • Compare protein detection with non-targeting control siRNA by Western blot

  • Verify reduction in band intensity at the expected molecular weight

• CRISPR knockout validation:

  • Generate RNASEH2B knockout cell lines or use commercially available ones

  • HeLa RNASEH2B CRISPR-knockouts have been demonstrated as effective controls

• Recombinant protein controls:

  • Overexpress tagged RNASEH2B protein (MYC-tagged overexpression systems have been validated)

  • Verify antibody detection of both endogenous and overexpressed protein

• Functional assays:

  • Confirm loss of RNase H2 activity in RNASEH2A/B knockout samples using FRET-based fluorescent substrate release assays

  • Increased genomic DNA fragmentation after RNase H2 treatment and alkaline gel electrophoresis indicates embedded ribonucleotides in knockout cells

• Multiple antibody comparison:

  • Use antibodies targeting different epitopes of RNASEH2B

  • Compare staining patterns across different applications (WB, IHC, IF)

What are common issues when using RNASEH2B antibodies and how can they be addressed?

How can RNASEH2B antibodies be used to study RNA:DNA hybrid dynamics?

RNASEH2B antibodies enable several advanced approaches to study RNA:DNA hybrid dynamics:

• Immunoprecipitation-based methods:

  • Co-immunoprecipitation (Co-IP) using RNASEH2B antibodies to identify interaction partners in the RNase H2 complex

  • Chromatin immunoprecipitation (ChIP) to map RNASEH2B binding sites on chromatin

• Combined antibody approaches:

  • Double immunostaining with S9.6 antibody (detects RNA:DNA hybrids) and RNASEH2B antibody

  • This approach reveals the relationship between RNASEH2B localization and RNA:DNA hybrid accumulation

• Functional studies:

  • Combine RNASEH2B immunodetection with slot blot analysis using S9.6 antibody to correlate RNASEH2B levels with global RNA:DNA hybrid signals

  • Inducible RNASEH2B overexpression systems show that RNASEH2B overexpression alone can increase RNA:DNA hybrid signals, but prevents further increases after drug treatments that induce replication stress

• Cell cycle studies:

  • Double staining with cell cycle markers (e.g., PCNA) and RNASEH2B

  • RNASEH2B contains a functional PIP domain that allows interaction with PCNA

These approaches have revealed that RNASEH2B overexpression unexpectedly increases global RNA:DNA hybrid levels but prevents further increases induced by replication stress agents like camptothecin or hydroxyurea .

How can RNASEH2B antibodies be applied in cancer research?

RNASEH2B antibodies are increasingly important in cancer research with several applications:

• Biomarker development:

  • IHC screening for RNASEH2B protein loss in tumor samples

  • In leiomyosarcoma, RNase H2 loss was found in 11.5% of cases, higher than across all tumors (3.8%)

  • Uterine leiomyosarcoma shows higher rates of RNase H2 deficiency (21%) compared to other cancers

• Therapeutic response prediction:

  • RNASEH2B loss is unrelated to homologous recombination deficiency (HRD) but preclinically sensitizes to PARP inhibition

  • IHC detection of RNASEH2B loss could identify candidates for PARP inhibitor therapy

  • In prostate cancer studies, patients with RNASEH2B protein loss are being evaluated for PARP inhibitor response

• Tumor heterogeneity analysis:

  • Both homogeneous and heterogeneous RNASEH2B protein loss patterns are observed in cancer samples

  • Intra-tumor heterogeneity of RNASEH2B expression may have implications for treatment resistance

• Correlation with genomic alterations:

  • RNASEH2B IHC has 93% sensitivity and 71% specificity for detecting RNASEH2B homozygous deletions in uterine leiomyosarcoma

Research has shown that RNASEH2B loss frequently co-occurs with RB1 protein loss in prostate cancer, which may decrease PARP inhibitor sensitivity , demonstrating the complexity of biomarker development.

How should researchers interpret contradictory results between RNASEH2B protein levels and RNA:DNA hybrid levels?

One of the most intriguing findings in recent RNASEH2B research is the seemingly paradoxical relationship between RNASEH2B levels and RNA:DNA hybrids:

These contradictions highlight the complex regulation of RNA:DNA hybrid metabolism and suggest that simple models of enzyme-substrate relationships may be insufficient.

What factors should be considered when analyzing RNASEH2B expression in different tissue types?

When analyzing RNASEH2B expression across tissues:

• Tissue-specific considerations:

  • Bone biopsies: EDTA decalcification can artificially reduce RNASEH2B staining

  • Brain tissue: RNASEH2B mutations are associated with Aicardi-Goutières syndrome, affecting brain function

  • Cancer tissues: Expression is often heterogeneous and may correlate with genomic instability markers

• Technical factors impacting analysis:

  • Fixation time: Standardize for consistent results

  • Antigen retrieval: Critical for nuclear antigens like RNASEH2B

  • Scoring systems: Nuclear H-score is recommended as RNASEH2B is predominantly nuclear

  • Digital vs. visual scoring: Both methods correlate well with HALO digital analysis systems

• Biological variables to consider:

  • Cell proliferation state: Replication stress affects RNASEH2B levels

  • Treatment history: Chemotherapy drugs can induce RNASEH2B upregulation

  • Oncogene activation: HRAS activation increases RNASEH2B levels

  • Heterogeneity: Both intra- and inter-tumor heterogeneity in RNASEH2B expression are common

• Comparative analysis frameworks:

  • Use multiple internal controls within samples

  • Consider parallel RNASEH2A and RNASEH2C staining as all three subunits form the functional complex

  • Include normal adjacent tissue when analyzing tumor samples

This complex landscape explains why RNASEH2B expression patterns must be interpreted within appropriate tissue-specific and experimental contexts.

How might RNASEH2B antibodies be used to study the relationship between RNase H2 and innate immunity?

RNASEH2B antibodies offer powerful tools to explore emerging connections between RNase H2 and innate immunity:

• Investigation approaches:

  • Co-localization studies: Combine RNASEH2B immunostaining with markers of cytosolic DNA sensors (cGAS, STING)

  • Proximity ligation assays: Detect interactions between RNASEH2B and innate immune components

  • Correlation analyses: Link RNASEH2B levels with activation of TBK1 (phospho-S172), a key regulator of innate immunity

• Research questions to address:

  • How does RNASEH2B deficiency affect cytoplasmic nucleic acid accumulation?

  • Does RNASEH2B overexpression modulate the induction of interferon-stimulated genes (ISGs)?

  • What is the relationship between micronuclei formation, RNASEH2B levels, and innate immune activation?

• Experimental systems:

  • RNASEH2B-overexpressing cells treated with replication stress agents

  • RNASEH2B-deficient models of Aicardi-Goutières syndrome

  • Cancer cells with varying RNASEH2B expression levels exposed to immunotherapy

Preliminary research has shown that RNASEH2B overexpression can affect TBK1 phosphorylation , suggesting a direct link to innate immune signaling pathways. RNASEH2B's role in preventing inappropriate immune activation by removing unnecessary DNA fragments is particularly relevant to Aicardi-Goutières syndrome pathophysiology .

What are the emerging techniques combining RNASEH2B antibodies with other advanced methodologies?

Several cutting-edge approaches are being developed:

• Single-cell applications:

  • Single-cell proteomics with RNASEH2B antibodies to assess heterogeneity

  • Combined single-nucleus RNA-Seq with antibody-based protein detection for multi-omics analysis

  • Spatial transcriptomics with RNASEH2B immunofluorescence for location-specific correlation

• Live-cell imaging techniques:

  • CRISPR-based tagging of endogenous RNASEH2B combined with antibody-based verification

  • Fluorescent nanobodies derived from validated RNASEH2B antibodies for real-time tracking

  • FRET-based sensors to monitor RNase H2 complex assembly and activity

• Therapeutic development applications:

  • Using RNASEH2B antibodies for companion diagnostics in clinical trials of PARP inhibitors

  • Combining RNASEH2B immunodetection with other DDR markers for comprehensive DNA repair profiling

  • Developing RNASEH2B antibody-drug conjugates for targeted therapy in cancers overexpressing RNASEH2B

• In situ interaction studies:

  • Antibody-based RNASEH2B detection combined with PCNA visualization to study interactions at replication forks

  • Proximity ligation assays to identify novel RNASEH2B interaction partners in different cellular compartments

These emerging techniques will help resolve important questions about RNASEH2B's dynamic roles in normal physiology and disease states, particularly in cancer and neurological disorders.