Recombinant Human Putative uncharacterized protein C14orf132 (C14orf132)

What are the key specifications of commercially available recombinant C14orf132 protein?

Recombinant Human Putative uncharacterized protein C14orf132 is available commercially with the following specifications:

The recombinant protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol (final concentration) added for long-term storage at -20°C or -80°C . For short-term use, working aliquots can be stored at 4°C for up to one week, though repeated freezing and thawing is not recommended as it may compromise protein stability and functionality .

What experimental methods are recommended for studying C14orf132 expression patterns?

For investigating C14orf132 expression patterns, multiple complementary approaches are recommended to enhance reliability:

RNA sequencing (RNA-seq) has proven effective for detecting differential expression of C14orf132 between study groups, as demonstrated in a family study of ELBW children where C14orf132 RNA was undetectable in affected children while present in parental samples . When implementing RNA-seq for C14orf132 detection, recommended sequencing depth should be sufficient to detect low-abundance transcripts (minimum 30 million reads per sample).

Quantitative reverse transcription PCR (qRT-PCR) using TaqMan assay has successfully validated RNA-seq findings related to C14orf132 expression differences . When designing qRT-PCR experiments, appropriate reference genes should be carefully selected based on the tissue type being analyzed to ensure accurate normalization.

For both methods, proper experimental controls are crucial, particularly when investigating familial patterns of expression. The case study examining C14orf132 in ELBW children demonstrated how control families (with appropriately sized newborns) should be included to determine whether expression differences are specific to the condition being studied or represent normal variation .

What approaches should be used to investigate the functional role of C14orf132 as a long non-coding RNA?

Investigating the functional role of C14orf132 as a lincRNA requires specialized experimental approaches that differ from those used for protein-coding genes:

RNA-protein interaction studies, including RNA immunoprecipitation (RIP) and cross-linking immunoprecipitation (CLIP) techniques, should be employed to identify protein binding partners of C14orf132, providing insights into its molecular mechanisms. These methods would help determine whether C14orf132 functions by interacting with transcription factors, chromatin modifiers, or other regulatory proteins.

Knockdown and overexpression experiments using siRNA/shRNA for knockdown and lentiviral vectors for overexpression in relevant cell lines (particularly neuronal cells, given C14orf132's high brain expression) would help identify downstream genes and pathways affected . RNA-seq analysis of knockdown/overexpression models could reveal co-regulated genes and potential functional networks.

Subcellular localization studies using fractionation techniques followed by qRT-PCR or RNA fluorescence in situ hybridization (RNA-FISH) would determine whether C14orf132 functions in the nucleus (suggesting transcriptional regulation) or cytoplasm (suggesting post-transcriptional mechanisms).

CRISPR-Cas9 genome editing could be used to create cellular or animal models with C14orf132 deletion or mutation to observe phenotypic changes, particularly focusing on developmental processes given its potential role in pre- and postnatal development .

How might researchers investigate the potential link between C14orf132 and extremely low birth weight?

The reported association between C14orf132 downregulation and extremely low birth weight warrants thorough investigation through multiple approaches:

Case-control studies comparing C14orf132 expression in placental tissue, cord blood, and maternal blood between ELBW cases and appropriate controls (both normal birth weight and small for gestational age without extreme prematurity) would help establish whether expression differences are specific to ELBW or related to growth restriction in general. Previous research found expression differences were unique to a specific ELBW family and not present in small for gestational age (SGA) control families .

Epigenetic analysis of the C14orf132 locus, including DNA methylation and histone modification profiling, should be performed since copy number variation (CNV) analysis did not reveal genomic aberrations in the C14orf132 region in ELBW children, suggesting potential epigenetic regulation mechanisms .

Temporal expression profiling throughout pregnancy in animal models would provide insights into C14orf132's role in developmental processes. Coupled with interventional studies modulating C14orf132 expression, this approach could establish causative relationships with developmental outcomes.

Multi-omics integration combining transcriptomics, epigenomics, and phenotypic data from larger cohorts of ELBW cases would help identify whether C14orf132 downregulation is part of broader regulatory networks affecting pre- and postnatal development .

What experimental design considerations are important when investigating C14orf132 in disease contexts?

Given C14orf132's reported downregulation in cancer and potential developmental roles, several experimental design considerations are critical:

Tissue specificity must be carefully addressed since C14orf132 shows differential expression across tissues, with highest expression in brain . Experiments should include appropriate tissue controls and account for tissue-specific regulatory mechanisms when investigating disease associations.

Cell type heterogeneity within tissues should be considered when analyzing C14orf132 expression, as bulk tissue measurements may mask cell type-specific patterns. Single-cell RNA sequencing would be valuable for characterizing expression at cellular resolution, particularly in developmental contexts or heterogeneous tumors.

Temporal dynamics in expression should be captured through time-course experiments, especially when studying developmental processes where C14orf132 may play stage-specific roles. This is particularly relevant given its potential association with pre- and early postnatal development .

The appropriate model system selection is crucial since lincRNA functions can be species-specific with poor conservation. Human cell lines, organoids, or primary tissues should be prioritized over animal models, though the latter may still provide valuable insights into developmental processes if C14orf132 has conserved orthologs.

How should researchers address data inconsistencies when studying low-abundance transcripts like C14orf132?

When working with low-abundance transcripts like C14orf132, data inconsistencies require systematic approaches:

Technical validation across multiple platforms is essential. As demonstrated in the ELBW study, findings from RNA-seq were validated using qRT-PCR with TaqMan assay to confirm expression differences observed in the original analysis . This multi-method approach increases confidence in results, particularly for transcripts expressed at low levels.

Statistical approaches for low-abundance transcripts should be optimized, including appropriate normalization methods (such as TMM or quantile normalization) and variance stabilization techniques to account for the high variability typical of low-count data. The ELBW study employed statistical significance thresholds including false discovery rate (FDR) calculations to minimize false positives .

Biological replicates should be increased beyond standard numbers when studying low-abundance transcripts to enhance statistical power. For C14orf132 specifically, inclusion of both biological and technical replicates would help distinguish true biological variation from technical noise.

RNA quality assessment is particularly important for low-abundance transcripts as degradation disproportionately affects detection of rare RNAs. Rigorous quality control measures including RNA integrity number (RIN) assessment should be implemented, with threshold values ≥8 recommended for studies of low-abundance lincRNAs.

What downstream analysis approaches are recommended after identifying differential expression of C14orf132?

When C14orf132 shows differential expression in experimental or clinical samples, several downstream analyses can provide functional insights:

Gene co-expression network analysis can identify genes whose expression patterns correlate with C14orf132 across samples, potentially revealing functional relationships and regulatory networks. This approach is particularly valuable for uncharacterized genes like C14orf132, as it provides functional guilt-by-association .

Pathway enrichment analysis of co-expressed genes can highlight biological processes potentially influenced by C14orf132. Standard approaches include Gene Ontology (GO) analysis for molecular functions, cellular components, and biological processes, as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis .

Integration with clinical data is essential to establish phenotypic correlations, as exemplified in the ELBW study where C14orf132 expression differences were correlated with specific clinical phenotypes . For broader applicability, researchers should examine correlations with additional phenotypes using databases like NCBI PheGenI, NCBI ClinVar, and NCBI dbVar .

Comparative analysis across species can provide evolutionary context, though this may be limited for lincRNAs which often exhibit poor sequence conservation. Structure conservation analysis may be more informative for functional prediction of lincRNAs like C14orf132.

What are the recommended protocols for handling and reconstituting recombinant C14orf132 protein for experimental use?

When working with recombinant C14orf132 protein, the following handling and reconstitution protocols are recommended:

Pre-reconstitution preparation should include brief centrifugation of the vial prior to opening to ensure all content is collected at the bottom . All subsequent steps should be performed under sterile conditions to prevent contamination.

Reconstitution should be performed using deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL . For optimal solubilization, the solution should be gently mixed rather than vortexed to prevent protein denaturation. Temperature during reconstitution should be maintained at room temperature or 4°C depending on the specific experimental requirements.

For long-term storage, addition of glycerol to a final concentration of 5-50% is recommended, with 50% being the standard protocol for maximal stability . The solution should then be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C/-80°C. Under these conditions, reconstituted protein maintains stability for approximately 6 months, while lyophilized forms remain stable for up to 12 months .

For working solutions, storage at 4°C is suitable for up to one week, though activity should be validated periodically if stored for extended periods . Repeated freezing and thawing must be avoided as this significantly impacts protein stability and function.

How should researchers design experiments to investigate potential epigenetic regulation of C14orf132?

Given the evidence suggesting epigenetic regulation of C14orf132 expression, particularly in developmental contexts, the following experimental design approaches are recommended:

DNA methylation analysis of the C14orf132 promoter region should be performed using bisulfite sequencing or methylation-specific PCR. This is particularly relevant since the ELBW study found no copy number variations in the C14orf132 region despite significant expression differences, suggesting potential epigenetic mechanisms .

Chromatin accessibility assays including ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) would identify open chromatin regions potentially involved in C14orf132 regulation. Comparing these profiles between samples with different C14orf132 expression levels (e.g., parental samples versus ELBW children) could reveal regulatory mechanisms .

Transcription factor binding site analysis at the C14orf132 locus, using both in silico prediction and experimental validation through ChIP, would identify potential upstream regulators. Correlation analysis between expression levels of identified transcription factors and C14orf132 across relevant samples would strengthen evidence for regulatory relationships.