Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform

<p>Abstract</p> <p>Background</p> <p>The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel^v1(1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.</p> <p>Methods</p> <p>Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance <it>t</it>-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).</p> <p>Results</p> <p>Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, <it>erythroblastic leukemia viral oncogene homolog 2 </it>(<it>ERBB2</it>) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: <it>topoisomerase II alpha </it>(<it>TOP2A</it>), <it>cyclin a2 </it>(<it>CCNA2</it>), <it>v-fos fbj murine osteosarcoma viral oncogene homolog </it>(<it>FOS</it>), <it>wingless-type mmtv integration site family, member 5a </it>(<it>WNT5A</it>), <it>growth factor receptor-bound protein </it><it>7 </it>(<it>GRB7</it>), <it>cell division cycle 2 </it>(<it>CDC2</it>), <it>and baculoviral iap repeat-containing protein 5 </it>(<it>BIRC5</it>). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around <it>v-myc avian myelocytomatosis viral oncogene homolog </it>(<it>MYC</it>), <it>tumor protein p53 </it>(<it>TP53</it>), and <it>estrogen receptor α </it>(<it>ESR1</it>). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through <it>MYC</it>, <it>TP53</it>, and <it>ESR1</it>.</p> <p>Conclusions</p> <p>The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.</p

A database application for pre-processing, storage and comparison of mass spectra derived from patients and controls.

BACKGROUND: Statistical comparison of peptide profiles in biomarker discovery requires fast, user-friendly software for high throughput data analysis. Important features are flexibility in changing input variables and statistical analysis of peptides that are differentially expressed between patient and control groups. In addition, integration the mass spectrometry data with the results of other experiments, such as microarray analysis, and information from other databases requires a central storage of the profile matrix, where protein id's can be added to peptide masses of interest. RESULTS: A new database application is presented, to detect and identify significantly differentially expressed peptides in peptide profiles obtained from body fluids of patient and control groups. The presented modular software is capable of central storage of mass spectra and results in fast analysis. The software architecture consists of 4 pillars, 1) a Graphical User Interface written in Java, 2) a MySQL database, which contains all metadata, such as experiment numbers and sample codes, 3) a FTP (File Transport Protocol) server to store all raw mass spectrometry files and processed data, and 4) the software package R, which is used for modular statistical calculations, such as the Wilcoxon-Mann-Whitney rank sum test. Statistic analysis by the Wilcoxon-Mann-Whitney test in R demonstrates that peptide-profiles of two patient groups 1) breast cancer patients with leptomeningeal metastases and 2) prostate cancer patients in end stage disease can be distinguished from those of control groups. CONCLUSION: The database application is capable to distinguish patient Matrix Assisted Laser Desorption Ionization (MALDI-TOF) peptide profiles from control groups using large size datasets. The modular architecture of the application makes it possible to adapt the application to handle also large sized data from MS/MS- and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry experiments. It is expected that the higher resolution and mass accuracy of the FT-ICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles

Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive or negative regulators of cell proliferation and apoptosis. Here, we evaluated miRNA expression in formalin fixed, paraffin embedded (FFPE) samples and fresh frozen (FF) samples using a high throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms using the same samples. We obtained a highly correlated Ct values between multiplex and single-plex RT reactions using standard qPCR assays for miRNA expression. For the same samples, the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left shift towards lower Ct values compared to those observed by standard TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs at a wide range of expression values using a single 96-multiplexing RT reaction in either FFPE or FF samples. Comparison of miRNAs expression values measured by microfluidic technology with those obtained by other array and Next Generation sequencing platforms showed positive concordance using the same samples but revealed significant differences for a large fraction of miRNA targets. The qPCRarray based microfluidic technology can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. This approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform while achieving much higher throughput at lower sample input and reagent usage. It is a rapid, cost effective, customizable array platform for miRNA expression profiling and validation. However, comparison of miRNA expression using different platforms requires caution and the use of multiple platforms

Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods.

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS

The structure of 5-aza-2,8-dioxa-1-stanna(II)bicyclo[3.3.0]octane

From the1H,13C, and119Sn NMR spectra at different temperatures, it is concluded that the 5-alkyl-5-aza-2,8-dioxa-1-stanna(II)bicyclo[3.3.0]octanes dimerize in nonpolar solvents. The barrier for dimerization is lower (G =40 kJ/mol) than the barrier for simultaneous dissociation-inversion (G =70 kJ/mol). The dimers studied are sterically uniform

1,3,2,5-dioxadiphosphorinanes - 6-membered Ring Compounds Containing 2 Different Phosphorus Atoms

The reaction of bis(hydroxymethyl)phenylphosphane with tert-butyldichlorophosphane leads to the formation of cis-2-tert-butyl-5-phenyl-1,3,2,5-dioxadiphosphorinane (1). The cis configuration was definitively proven by the X-ray diffraction study of the 2, 5-dithioxo derivative (2), which was obtained from (1) by a stereoselective reaction. The diselenoxo compound (3) was also prepared. These three new compounds were subjected to a complete NMR study (1H,13C,31P, and77Se). © 1985 Plenum Publishing Corporation.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

8-membered Heterocycles Containing 2 Phosphorus Atoms - X-ray-diffraction and Nuclear-magnetic-resonance Studies of 2,6-dimethyl-1,3-dioxa-2,6-diphosphacyclo-octane 2,6-disulfides and 2,6-diselenides

trans- and cis-2,6-dimethyl-1,3-dioxa-2,6-diphosphacyclo-octane 2,6-disulphides (1) and (2), and trans- and cis-2,6-diselenides (3) and (4), have been investigated by means of 1H (2D, J-resolved), 13C, and 31P n.m.r. spectroscopy and X-ray diffraction. In the solid state the cis-isomers adopt a crown conformation and the trans-isomers a boat-chair conformation. The orientations of P-X (X = S or Se) and P-Me bonds are quasi-axial and quasi-equatorial, respectively. Room-temperature n.m.r. data can be interpreted on the basis of the X-ray data. A fast enantiomerization between two boat-chair conformations of opposite chirality characterizes the trans-isomers, whereas the cis-isomers exist mainly in a crown conformation similar to that observed in the crystal.SCOPUS: ar.jinfo:eu-repo/semantics/publishe