Stability and reproducibility of proteomic profiles measured with an aptamer-based platform.

The feasibility of SOMAscan, a multiplex, high sensitivity proteomics platform, for use in studies using archived plasma samples has not yet been assessed. We quantified 1,305 proteins from plasma samples donated by 16 Nurses' Health Study (NHS) participants, 40 NHSII participants, and 12 local volunteers. We assessed assay reproducibility using coefficients of variation (CV) from duplicate samples and intra-class correlation coefficients (ICC) and Spearman correlation coefficients (r) of samples processed (i.e., centrifuged and aliquoted into separate components) immediately, 24, and 48 hours after collection, as well as those of samples collected from the same individuals 1 year apart. CVs were <20% for 99% of proteins overall and <10% for 92% of proteins in heparin samples compared to 66% for EDTA samples. We observed ICC or Spearman r (comparing immediate vs. 24-hour delayed processing) ≥0.75 for 61% of proteins, with some variation by anticoagulant (56% for heparin and 70% for EDTA) and protein class (ranging from 49% among kinases to 83% among hormones). Within-person stability over 1 year was good (ICC or Spearman r ≥ 0.4) for 91% of proteins. These results demonstrate the feasibility of SOMAscan for analyses of archived plasma samples.

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.

The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community.

Reproducibility of Circulating MicroRNAs in Stored Plasma Samples.

BACKGROUND: Most studies of microRNA (miRNA) and disease have examined tissue-specific expression in limited numbers of samples. The presence of circulating miRNAs in plasma samples provides the opportunity to examine prospective associations between miRNA expression and disease in initially healthy individuals. However, little data exist on the reproducibility of miRNAs in stored plasma. METHODS: We used Real-Time PCR to measure 61 pre-selected microRNA candidates in stored plasma. Coefficients of variation (CVs) were used to assess inter-assay reliability (n = 15) and within-person stability over one year (n = 80). Intraclass correlation coefficients (ICCs) and polychoric correlation coefficients were used to assess within-person stability and delayed processing reproducibility (whole blood stored at 4°C for 0, 24 and 48 hours; n = 12 samples). RESULTS: Of 61 selected miRNAs, 23 were detected in at least 50% of samples and had average CVs below 20% for inter-assay reproducibility and 31 for delayed processing reproducibility. Ten miRNAs were detected in at least 50% of samples, had average CVs below 20% and had ICCs above 0.4 for within-person stability over 1-2 years, six of which satisfied criteria for both interassay reproducibility and short-term within-person stability (miR-17-5p, -191-5p, -26a-5p, -27b-3p, -320a, and -375) and two all three types of reproducibility (miR-27b-3p and -26a-5p). However, many miRNAs with acceptable average CVs had high maximum CVs, most had low expression levels, and several had low ICCs with delayed processing. CONCLUSIONS: About a tenth of miRNAs plausibly related to chronic disease were reliably detected in stored samples of healthy adults.

Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation.

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female>male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.