Biomedical subjects

Steven Perrin

Publications and source records attributed to Steven Perrin.

5 recordsLinked to original sources

PubMed2006

A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies.

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and 'in-house' platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by quantitative real-time (QRT)-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent preprocessing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.

Chromosome Mapping↗

PubMed2006

Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes.

We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen.

Animals↗

PubMed2005

Lymphotoxin-beta receptor signaling is required for the homeostatic control of HEV differentiation and function.

The lymphotoxin axis is important for the maintenance of several specialized lymphoid microenvironments in secondary lymphoid tissue. Lymphoid-tissue architecture is highly plastic and requires continual homeostatic signaling to maintain its basal functional state. The cellularity of lymph nodes in adult mice was reduced by systemic blockade of lymphotoxin-beta receptor (LTbeta R) signaling with a soluble decoy receptor both in resting and reactive settings. This reduction in cellularity resulted from greatly impaired lymphocyte entry into lymph nodes due to decreased levels of peripheral lymph node addressing (PNAd) and MAdCAM on high endothelial venules (HEV). LTbeta R signaling was required to maintain normal levels of RNA expression of MAdCAM, and also of PNAd by regulating the expression of key enzymes and scaffold proteins required for its assembly. Thus, the homeostatic maintenance of functional HEV status in adult mice relies largely on LTbeta R signaling.

Animals↗

PubMed2005

Application of functional genomic technologies in a mouse model of retinal degeneration.

Generation of tissue-specific, normalized and subtracted cDNA libraries has the potential to characterize the expression of rare transcriptional units not represented on Affymetrix GeneChips. Initial sequence analysis of our murine cDNA clone collections showed that as much as 86, 45, and 30% of clones are not represented on the Affymetrix Mu11k, MG-U74, and MG-430 chip sets, respectively. A detailed study that compared EST sequences of a subtracted library generated from mouse retina to those of MG-430 consensus sequences was undertaken, using UniGene build 124 as the common reference. A set of 1111 nonredundant transcript regions, not represented on the commercial array, was identified. These clusters were used as the primary filter for analyzing a data set produced by assaying samples from the Pde6b(rd1) mouse model of retinal degeneration on a 12,325-feature retinal cDNA microarray. QRT-PCR validated eight unique transcripts identified by microarray. Seven of the transcripts showed retina-specific expression. Full-length cloning strategies were applied to two of the ESTs. The genes discovered by this approach are the full-length mouse homologue of guanylate cyclase 2F (GUCY2F) and a carboxy-truncated splice variant of retinal S-antigen (SAG), known as regulators of the visual phototransduction G-protein-coupled receptor-mediated signaling pathway. These sequences have been assigned GenBank Accession Nos. and , respectively.

Animals↗

PubMed2002

Nondestructive quality control for microarray production.

The use of microarrays to monitor gene expression has become a standard research tool at both academic and industrial research institutions. Quality control of common printing defects during DNA deposition onto glass substrates is critical to maintaining data integrity and preventing the needless consumption of precious RNA, labeling reagents, and time. Here we demonstrate a nondestructive method for monitoring the quality of every spot on every chip of a microarray production run. We have identified many common manufacturing defects, while not perturbing the attachment of our oligonucleotide target to the substrate or altering further hybridization. This protocol is simple, fast, and inexpensive.

Carbocyanines↗