Recombinant Mouse Rhomboid-related protein 3 (Rhbdl3)

What is Mouse Rhomboid-related protein 3 (Rhbdl3) and what are its predicted functions?

Rhomboid-related protein 3 (Rhbdl3), also referred to as RHBDL4 or VRHO in certain databases, is a member of the rhomboid family of serine proteases. It is predicted to enable serine-type endopeptidase activity and is primarily involved in proteolysis . Structurally, it is characterized as an integral component of membrane, which aligns with the broader rhomboid family's role in regulated intramembrane proteolysis.

Functionally, mouse Rhbdl3 shares characteristics with its human ortholog RHBDL4, which has been identified as a promoter of endoplasmic reticulum-associated degradation (ERAD) of membrane proteins . Like other rhomboid proteases, it likely participates in various cellular processes including protein quality control, cellular signaling, and potentially developmental regulation through the selective cleavage of substrate proteins within the membrane environment.

Research methodologies to investigate its functions typically involve:

• Expression profiling across tissues

• Substrate identification through proteomics

• Loss-of-function studies via gene knockout or knockdown

• Gain-of-function experiments using recombinant protein expression systems

How does the structure of Rhbdl3 compare with other rhomboid family proteins?

The structure of mouse Rhbdl3 follows the characteristic architecture of rhomboid family proteins, featuring multiple transmembrane domains that anchor the protein within cellular membranes. The full-length mouse Rhbdl3 protein consists of 404 amino acids . When comparing Rhbdl3 with other rhomboid family members, several structural features become apparent:

• Transmembrane topology: Rhbdl3 contains multiple predicted transmembrane segments that form the core of the protein, with the catalytic serine residue located within one of these transmembrane domains.

• Catalytic machinery: Like other active rhomboid proteases, Rhbdl3 contains a catalytic dyad (serine and histidine) responsible for its proteolytic activity, distinguishing it from inactive rhomboid proteins (iRhoms).

• Substrate recognition regions: Specific extramembrane domains likely participate in substrate recognition and specificity.

Structural analysis approaches include:

• Sequence alignment with characterized rhomboid proteins

• Hydropathy profiling to predict transmembrane regions

• Three-dimensional structure prediction using tools like Phyre2, which have been employed to analyze splice variants of rhomboid proteins

• Comparative analysis with the bacterial rhomboid GlpG, which serves as a structural model for the family

What expression systems are available for producing recombinant mouse Rhbdl3?

Multiple expression systems have been developed for the production of recombinant mouse Rhbdl3, each with distinct advantages depending on the research application:

Methodological approach for expression in E. coli:

• Clone the Rhbdl3 coding sequence into a pET20b vector with C-terminal His-tag

• Transform into E. coli JM109 (DE3)

• Culture at 16°C in ampicillin-containing Terrific Broth (25 μg/ml)

• Induce expression with IPTG (typically 0.1-0.5 mM)

• Harvest cells and lyse using appropriate buffer systems

• Purify using nickel-NTA affinity chromatography

• Verify protein identity and purity by SDS-PAGE and immunoblotting with anti-rhomboid or anti-His antibodies

What is the role of alternative splicing in diversifying Rhbdl3 functionality, and how can splice variants be experimentally characterized?

Alternative splicing represents a significant mechanism for expanding the functional diversity of rhomboid proteins including Rhbdl3. Comparative genomic analyses across model organisms (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) have revealed robust usage of alternative splicing to diversify rhomboid protein structure . For rhomboid proteins, alternative splicing can affect various functional domains, potentially altering:

• Substrate specificity

• Subcellular localization

• Catalytic activity

• Protein-protein interaction networks

• Regulatory properties

Methodological approaches for characterizing Rhbdl3 splice variants include:

Computational analysis:

• Database mining of transcript variants (GenBank, Ensembl, UCSC Genome Browser)

• Sequence alignment using tools like Clustal Omega

• Prediction of structural changes using Phyre2 and visualization with PyMol

• Comparison with established structural models like bacterial rhomboid GlpG

Experimental validation:

• RT-PCR and qPCR to quantify splice variant expression in different tissues

• Recombinant expression of individual splice variants

• Functional assays:

  • Proteolytic activity assays using fluorogenic substrates

  • Transgenic expression in model systems

  • Additive-based assays where purified recombinant variants are added exogenously to cells (e.g., using amphotericin B to facilitate protein delivery)

• Subcellular localization studies using tagged variants and confocal microscopy

• Immunoblotting to assess expression and stability

For functional assessment, the amphotericin B-mediated protein delivery approach is particularly valuable:

• Incubate cells with 10 μg (at 1 μg/mL) of recombinant variant protein

• Include 1% (v/v) amphotericin B solution to create transmembrane channels

• Incubate for one hour before assessing functional outcomes

• Include appropriate controls with diluted elution buffer without recombinant protein

How does mouse Rhbdl3 interact with the ubiquitin-proteasome system, and what experimental approaches can elucidate these interactions?

Based on studies of the human ortholog RHBDL4, mouse Rhbdl3 likely participates in ER-associated degradation (ERAD) of membrane proteins and physically interacts with ubiquitin to facilitate its protease activities . This interaction represents a critical point of integration between proteolytic processing and protein quality control pathways.

The experimental strategy to investigate these interactions includes:

Biochemical approaches:

• Co-immunoprecipitation (Co-IP):

  • Express tagged Rhbdl3 in mammalian cells

  • Perform IP using anti-tag antibodies

  • Detect interacting ubiquitin or ubiquitinated proteins by immunoblotting

  • Include controls with catalytically inactive Rhbdl3 mutants

• Pull-down assays:

  • Use recombinant Rhbdl3 as bait

  • Incubate with cell lysates or purified ubiquitin

  • Analyze bound proteins by MS/MS to identify ubiquitin and ubiquitin-related proteins

• Ubiquitination assays:

  • Express Rhbdl3 with His-tagged ubiquitin

  • Purify ubiquitinated proteins under denaturing conditions

  • Detect Rhbdl3 by immunoblotting to assess its ubiquitination status

Functional approaches:

• Proteasome inhibition studies:

  • Treat cells expressing Rhbdl3 with proteasome inhibitors (MG132, bortezomib)

  • Monitor changes in substrate levels and Rhbdl3 activity

• Ubiquitin binding domain mutations:

  • Identify putative ubiquitin-interacting motifs in Rhbdl3

  • Generate point mutations in these regions

  • Assess effects on substrate degradation and ERAD function

• Proximity labeling techniques:

  • Fuse Rhbdl3 to BioID or APEX2

  • Express in cells and activate labeling

  • Identify proximal proteins, focusing on ubiquitin pathway components

The interplay between Rhbdl3 and the ubiquitin-proteasome system likely influences substrate selection, proteolytic efficiency, and the regulation of Rhbdl3 itself. This represents an important area for investigation in understanding the physiological roles of this protein.

What are the known or predicted substrates of mouse Rhbdl3, and what methodologies are most effective for substrate identification?

The identification of physiological substrates represents a significant challenge in rhomboid protease research, including for mouse Rhbdl3. While specific substrates for mouse Rhbdl3 are still being characterized, approaches for substrate identification combine computational prediction with experimental validation.

Computational prediction approaches:

• Sequence-based prediction:

  • Analysis of known rhomboid substrates to identify common motifs

  • Screening of membrane protein databases for these motifs

  • Evaluation of evolutionary conservation of potential cleavage sites

• Structural modeling:

  • Docking simulations between Rhbdl3 and candidate substrates

  • Assessment of accessibility of predicted cleavage sites within membrane environments

Experimental identification methodologies:

Validation of candidate substrates:

• In vitro cleavage assays:

  • Incubate purified recombinant Rhbdl3 with candidate substrate

  • Analyze cleavage products by SDS-PAGE and immunoblotting or MS

• Cell-based validation:

  • Co-express Rhbdl3 and tagged substrate in mammalian cells

  • Monitor substrate cleavage by immunoblotting

  • Compare with catalytically inactive Rhbdl3 mutant

  • Use Rhbdl3 knockdown or knockout to assess effects on endogenous substrate processing

• Cleavage site determination:

  • Edman sequencing or MS analysis of cleavage products

  • Site-directed mutagenesis of predicted cleavage sites

  • Assessment of cleavage efficiency with mutated substrates

This multi-faceted approach allows for comprehensive identification and validation of physiological Rhbdl3 substrates, providing insights into its cellular functions.

What are the optimal conditions for expression and purification of recombinant mouse Rhbdl3 for structural and functional studies?

Optimizing the expression and purification of recombinant mouse Rhbdl3 requires careful consideration of several parameters to obtain functional protein suitable for structural and functional analyses. Based on established protocols for rhomboid proteins, the following conditions are recommended:

Expression systems and conditions:

Purification protocol for E. coli-expressed Rhbdl3:

• Cell lysis:

  • Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1% DDM or other suitable detergent, protease inhibitors)

  • Sonicate or use French press for cell disruption

  • Centrifuge at 20,000 × g for 30 minutes to remove debris

• Affinity purification:

  • Apply cleared lysate to Ni-NTA resin equilibrated with washing buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.1% DDM)

  • Wash extensively to remove non-specifically bound proteins

  • Elute with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole, 0.1% DDM)

• Further purification:

  • Size exclusion chromatography using Superdex 200 in buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 0.05% DDM

  • For highest purity, consider ion exchange chromatography as an intermediate step

• Quality control:

  • SDS-PAGE and western blotting to assess purity and identity

  • Activity assays using fluorogenic peptide substrates

  • Circular dichroism to assess secondary structure

Critical considerations:

• Detergent selection is crucial for maintaining Rhbdl3 in a native-like membrane environment; screen multiple detergents (DDM, LMNG, GDN) for optimal activity retention

• Addition of lipids (E. coli polar lipids or specific phospholipids) may enhance stability and activity

• Avoid freeze-thaw cycles; store purified protein at 4°C for short-term or flash-freeze in small aliquots for long-term storage

• For structural studies, consider using nanodiscs or amphipols as alternatives to detergent micelles

These optimized conditions should yield recombinant mouse Rhbdl3 with >70-80% purity suitable for most functional and preliminary structural studies .

How can researchers effectively validate the activity of recombinant mouse Rhbdl3 in various experimental systems?

Validating the activity of recombinant mouse Rhbdl3 is essential to ensure that the protein retains its native functionality after expression and purification. Multiple complementary approaches can be employed across different experimental systems:

In vitro activity assays:

• Fluorogenic peptide substrates:

  • Design peptides containing a fluorophore and quencher separated by a sequence resembling known rhomboid cleavage sites

  • Incubate with purified Rhbdl3 and monitor fluorescence increase over time

  • Include controls with heat-inactivated enzyme and known inhibitors (e.g., DCI, isocoumarin derivatives)

  • Determine kinetic parameters (Km, kcat) for substrate hydrolysis

• Protein substrate cleavage:

  • Incubate recombinant Rhbdl3 with candidate protein substrates

  • Analyze reaction products by SDS-PAGE and immunoblotting

  • Confirm cleavage site by mass spectrometry

  • Compare activity against catalytically inactive mutant (typically serine to alanine mutation in the active site)

Cell-based validation:

• Reconstitution in proteoliposomes:

  • Incorporate purified Rhbdl3 into liposomes

  • Add fluorescently labeled substrate

  • Monitor cleavage by fluorescence or gel-based assays

• Cell-free translation systems:

  • Express Rhbdl3 in microsomal membranes

  • Co-express with substrate proteins

  • Assess cleavage by autoradiography or immunoblotting

• Mammalian cell expression:

  • Transfect cells with Rhbdl3 expression constructs

  • Monitor effects on endogenous or co-expressed substrates

  • Compare wild-type to catalytically inactive mutants

Whole-cell assays with exogenously added protein:

• Amphotericin B-mediated protein delivery:

  • Incubate cells with 10 μg/mL purified Rhbdl3 in the presence of 1% (v/v) amphotericin B

  • Include controls with buffer alone

  • Assess biological effects (e.g., changes in specific cellular pathways)

Functional complementation:

• Yeast-based assays:

  • Express mouse Rhbdl3 in yeast strains lacking endogenous rhomboid proteases

  • Assess rescue of phenotypes associated with rhomboid deficiency

  • Monitor effects on known yeast rhomboid substrates (e.g., effect on mitochondrial protein Mgm1)

Activity validation metrics:

These multi-level validation approaches ensure that the recombinant mouse Rhbdl3 retains its native proteolytic activity and biological functionality across different experimental contexts.

What are the most effective methods for studying protein-protein interactions involving mouse Rhbdl3?

Understanding the protein-protein interaction network of mouse Rhbdl3 is crucial for elucidating its biological functions, especially considering its interaction with ubiquitin and potential role in ER-associated degradation . Multiple complementary approaches can be employed to comprehensively map these interactions:

In vitro interaction studies:

• Pull-down assays:

  • Immobilize purified recombinant Rhbdl3 (with affinity tag) on appropriate resin

  • Incubate with cell lysates or purified candidate interacting proteins

  • Wash to remove non-specific binding

  • Elute and analyze bound proteins by immunoblotting or mass spectrometry

  • Include negative controls (unrelated protein or buffer)

• Surface Plasmon Resonance (SPR):

  • Immobilize Rhbdl3 on sensor chip

  • Flow candidate interacting proteins over the surface

  • Measure binding kinetics (kon, koff) and affinity (KD)

  • Validate interactions with reversed orientation (immobilize partner, flow Rhbdl3)

• Microscale Thermophoresis (MST):

  • Label Rhbdl3 with fluorescent dye

  • Measure thermophoretic movement in presence of increasing concentrations of interacting partner

  • Calculate binding affinity from dose-response curve

Cell-based interaction studies:

• Co-immunoprecipitation (Co-IP):

  • Express tagged Rhbdl3 in mammalian cells

  • Lyse cells in mild detergent buffer preserving protein complexes

  • Immunoprecipitate Rhbdl3 using tag-specific antibodies

  • Analyze co-precipitated proteins by immunoblotting or mass spectrometry

  • Include controls with unrelated tagged protein

• Proximity-based labeling:

  • Fuse Rhbdl3 to a proximity labeling enzyme (BioID, APEX2, TurboID)

  • Express in relevant cell type and activate labeling

  • Purify biotinylated proteins

  • Identify proximal proteins by mass spectrometry

  • Compare results with control constructs (e.g., inactive Rhbdl3 mutant)

• Fluorescence-based methods:

  • Förster Resonance Energy Transfer (FRET):

    • Express Rhbdl3 fused to donor fluorophore

    • Express candidate interacting protein fused to acceptor fluorophore

    • Measure energy transfer as indicator of protein proximity

  • Bimolecular Fluorescence Complementation (BiFC):

    • Fuse Rhbdl3 and candidate partner to complementary fragments of fluorescent protein

    • Reconstitution of fluorescence indicates interaction

    • Visualize by confocal microscopy or quantify by flow cytometry

Unbiased screening approaches:

• Yeast two-hybrid (Y2H):

  • Create fusion of Rhbdl3 (or domains) with DNA-binding domain

  • Screen against prey library fused to activation domain

  • Select for reporter gene activation

  • Validate hits by secondary assays

• Mammalian membrane two-hybrid:

  • Adaptation of Y2H for membrane proteins

  • Better suited for Rhbdl3 as an integral membrane protein

• Protein fragment complementation assays (PCA):

  • Split-ubiquitin system for membrane proteins

  • Reconstitution of reporter activity upon interaction

Data analysis and validation:

When investigating Rhbdl3 interactions, special consideration should be given to its membrane topology and the detergent environment used for extraction, as these factors significantly impact the preservation of physiologically relevant interactions.

How should researchers analyze and compare the functional differences between splice variants of mouse Rhbdl3?

Analyzing functional differences between splice variants of mouse Rhbdl3 requires a comprehensive approach that integrates structural prediction, biochemical characterization, and biological activity assessment. The strategy should account for potential alterations in various protein properties across the variants:

Structural and biochemical characterization:

• Sequence analysis and structural prediction:

  • Align splice variant sequences to identify regions of difference

  • Predict transmembrane topology changes using hydropathy analysis

  • Generate 3D structural models using tools like Phyre2 and visualize with PyMol

  • Compare to established rhomboid structures like bacterial GlpG

  • Quantify potential impact on active site geometry and substrate-binding regions

• Biochemical property assessment:

  • Express and purify each splice variant under identical conditions

  • Determine protein stability through thermal shift assays

  • Assess oligomerization state by size exclusion chromatography

  • Measure proteolytic activity against model substrates

  • Compare enzyme kinetics (Km, kcat, substrate specificity)

Functional comparison strategies:

• Cellular localization:

  • Express fluorescently tagged variants in mammalian cells

  • Perform co-localization studies with organelle markers

  • Quantify distribution patterns using image analysis software

  • Compare with immunostaining of endogenous protein when possible

• Substrate processing:

  • Co-express variants with known or candidate substrates

  • Measure substrate cleavage efficiency by immunoblotting

  • Determine if variants exhibit different substrate preferences

  • Use SILAC-based proteomics to identify differential substrate profiles

• Protein-protein interactions:

  • Perform comparative interactome analysis for each variant

  • Use proximity labeling or co-immunoprecipitation followed by mass spectrometry

  • Identify common and variant-specific interaction partners

  • Validate key differential interactions by direct binding assays

Biological activity assessment:

• Cell-based functional assays:

  • Express variants in relevant cell types

  • Measure impact on cellular processes (e.g., ER stress responses, protein quality control)

  • Assess effects on cell morphology, proliferation, or differentiation

  • Compare rescue efficiency in Rhbdl3-deficient cells

• Exogenous protein addition:

  • Use amphotericin B-mediated delivery to introduce purified variants into cells

  • Compare effects on cellular phenotypes and responses to stress conditions

  • Measure direct impact on potential physiological pathways

Data analysis and integration:

Case study approach:
For comprehensive analysis, researchers should conduct parallel characterization of all splice variants under identical conditions, generating quantitative metrics that allow direct comparison. The study by Powles et al. provides a model for this approach, where three splice variants of the plant rhomboid At1g74130 were systematically compared for their impact on mitochondrial morphology and function .

This multi-faceted approach allows researchers to build a comprehensive understanding of how alternative splicing modulates Rhbdl3 functionality, providing insights into the biological significance of this regulatory mechanism.

What statistical approaches are most appropriate for analyzing activity data from recombinant mouse Rhbdl3 experiments?

Analyzing activity data from recombinant mouse Rhbdl3 experiments requires careful selection of statistical methods appropriate to the experimental design, data distribution, and specific hypotheses being tested. Here are the recommended approaches for different experimental scenarios:

Statistical approaches for enzyme activity assays:

• Basic enzymatic activity comparison:

  • For comparing activity levels between wild-type and mutant Rhbdl3 or between different conditions:

    • Student's t-test (two groups) or ANOVA (multiple groups)

    • Non-parametric alternatives (Mann-Whitney or Kruskal-Wallis) if normality assumptions are violated

  • Report results as mean ± standard deviation or standard error with p-values

  • Include effect size measurements (Cohen's d or η²) to quantify magnitude of differences

• Enzyme kinetics analysis:

  • For Michaelis-Menten kinetics determination:

    • Non-linear regression to determine Km and Vmax values

    • Calculate 95% confidence intervals for each parameter

    • Compare parameters using extra sum-of-squares F test

  • For comparing kinetic parameters across conditions:

    • ANOVA with post-hoc tests for multiple comparisons

    • Analysis of covariance (ANCOVA) when controlling for covariates

Statistical approaches for cellular and functional assays:

• Dose-response experiments:

  • Non-linear regression to fit dose-response curves

  • Determination of EC50/IC50 values with confidence intervals

  • Statistical comparison of curves using extra sum-of-squares F test

  • Two-way ANOVA for comparing responses across multiple variants and concentrations

• Time-course experiments:

  • Repeated measures ANOVA or mixed-effects models

  • Area under the curve (AUC) analysis followed by appropriate comparative tests

  • Regression analysis to determine rate constants

  • Time-to-event analysis for threshold-crossing events

Statistical considerations for complex experiments:

Practical implementation guidelines:

• Sample size and power considerations:

  • Conduct a priori power analysis to determine required sample size

  • For typical enzymatic assays, aim for n ≥ 3 independent experiments with technical replicates

  • Report both biological and technical replication clearly

• Handling variability and outliers:

  • Assess normality using appropriate tests (Shapiro-Wilk, Kolmogorov-Smirnov)

  • Consider data transformations if assumptions are violated

  • Use robust statistical methods when appropriate

  • Establish clear criteria for outlier identification and handling

• Multiple testing correction:

  • When performing multiple comparisons, apply appropriate corrections:

    • Bonferroni correction (most conservative)

    • False Discovery Rate control (Benjamini-Hochberg procedure)

    • Tukey's or Dunnett's procedures for specific comparison patterns

• Reporting standards:

  • Report exact p-values rather than thresholds

  • Include measures of effect size alongside significance tests

  • Provide clear descriptions of statistical tests used

  • Present raw data where feasible (supplementary materials)

What are the common challenges in recombinant mouse Rhbdl3 expression and purification, and how can they be addressed?

Expressing and purifying functional recombinant mouse Rhbdl3 presents several challenges typical of integral membrane proteins, particularly those with proteolytic activity. Here are the most common issues researchers encounter and evidence-based solutions:

Challenge 1: Low expression levels

Challenge 2: Proteolytic activity issues

Challenge 3: Protein stability issues

Challenge 4: Purification challenges

Systematic optimization approach:

• Expression screening:

  • Test multiple expression systems in parallel (bacterial, insect, mammalian)

  • Evaluate different constructs (full-length vs. truncations)

  • Screen fusion tags and their positions

• Detergent optimization:

  • Perform systematic screening of detergent types

  • Test extraction efficiency and protein activity

  • Consider detergent exchange during purification

• Buffer optimization:

  • Vary pH, salt concentration, and additives

  • Perform thermal shift assays to identify stabilizing conditions

  • Test effects of specific lipids and cofactors

By systematically addressing these challenges with the suggested solutions, researchers can significantly improve the yield, purity, and activity of recombinant mouse Rhbdl3, enabling more reliable structural and functional studies.

What are the future research directions and emerging technologies relevant to mouse Rhbdl3 investigation?

The study of mouse Rhomboid-related protein 3 (Rhbdl3) represents an evolving field with significant opportunities for advancement through emerging technologies and new research directions. Based on current understanding and methodological innovations, several promising avenues for future investigation emerge:

Emerging structural biology approaches:

• Cryo-electron microscopy (cryo-EM):

  • Application to Rhbdl3 membrane protein complexes

  • Potential for visualization of substrate binding and catalytic intermediates

  • Reconstruction of dynamic conformational states during the catalytic cycle

  • Integration with computational modeling for complete structural understanding

• Integrative structural biology:

  • Combining X-ray crystallography, NMR, and cryo-EM data

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Single-particle analysis of different functional states

  • Correlation with molecular dynamics simulations

Advanced functional analysis methodologies:

• Genome engineering approaches:

  • CRISPR-Cas9 generation of endogenous tagged Rhbdl3 variants

  • Knock-in of specific splice variants to assess physiological roles

  • Creation of conditional knockout models for tissue-specific analysis

  • Base editing to introduce specific mutations at endogenous loci

• Spatiotemporal activity monitoring:

  • Development of FRET-based reporters for real-time monitoring of Rhbdl3 activity

  • Optogenetic control of Rhbdl3 expression or activity

  • Chemogenetic approaches for rapid and reversible functional modulation

  • Integration with live-cell imaging to correlate activity with cellular events

Systems biology integration:

• Multi-omics approach:

  • Integration of proteomics, transcriptomics, and metabolomics data

  • Network analysis to position Rhbdl3 within cellular signaling pathways

  • Identification of condition-specific regulation of Rhbdl3 activity

  • Machine learning approaches to predict contextual function

• Physiological and disease relevance:

  • Exploration of Rhbdl3 roles in development and tissue homeostasis

  • Investigation of connections to ER stress pathways and protein quality control

  • Assessment of potential roles in neurodegenerative disorders

  • Comparative analysis with human ortholog in disease models

Technological innovations with particular relevance:

Translational research directions:

• Development of specific inhibitors or modulators:

  • Structure-based design of Rhbdl3-specific compounds

  • Screening of natural product libraries for modulators

  • Exploration of potential therapeutic applications in protein misfolding disorders

• Biomarker potential:

  • Investigation of Rhbdl3 substrates as potential biomarkers

  • Analysis of splice variant expression in pathological conditions

  • Correlation with disease progression or treatment response