Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

Comparison of the expression patterns of genes coding for wheat gluten proteins and proteins involved in the secretory pathway in developing caryopses of wheat.

The synthesis of gluten proteins in the developing caryopsis of wheat is highly coordinated, with mRNAs for the various groups being detected from 11 days after anthesis, and the proteins from about 14 days. In contrast, the levels of transcripts for BiP, PDI and PPI are highest at earlier stages of development. The levels of transcripts for two small GTP binding proteins involved in the secretory pathway (Rab1 and Rab5) are also highest early in development, which is consistent with the retention of most of the gluten proteins within the ER to form protein bodies.

Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.

Physical and functional definition of the Drosophila Notch locus by P element transformation.

Notch is a developmentally regulated locus which controls the differentiation of various Drosophila tissues, among them the embryonic nervous system. Molecular analysis has suggested that Notch is defined by an approximately 40-kb transcription unit which is spliced into a 10.2-kb mRNA composed of nine exonic regions and coding for a 2703-amino acid long transmembrane protein that shows homology to the mammalian epidermal growth factor. Here, we define the 5' end of the transcription unit and determine the sequences deleted in a Notch mutation involving the 5' nontranscribed region. Using a Notch cosmid vector we demonstrate by P element-mediated transformation that all sequences necessary for Notch function are confined in an approximately 40-kb long genomic region. cDNA sequences are used to construct a 15-kb "minigene" which lacks most, but not all, introns and its functionality is also tested by P element transformation. We show that, unlike the cosmid vector which is capable of rescuing completely all Notch mutations, only certain phenotypes can be rescued by the "minigene." The functional implications of our findings are discussed.

The molecular genetics of the Notch locus in Drosophila melanogaster.

The Notch locus of Drosophila melanogaster profoundly affects differentiation of the central nervous system in the early embryo. Previous molecular studies suggested that the locus spans 40 kb of DNA and encodes a 10.5-kb poly(A)+ RNA. The results of genetic, cytogenetic, and molecular studies of newly induced and preexisting Notch alleles are reported. Molecular analysis of 5' flanking mutations defines a distal limit for the locus, and transcriptional activity of Notch in relation to that of flanking transcription units provides evidence regarding the proximal limit. Examination of a set of mutable alleles implicates a foldback transposable element as the basis of chromosomal instability, and shows that insertion sequences within the locus do not necessarily result in a mutant phenotype. The results are discussed with regard to existing developmental and genetic analyses, and a molecular model is proposed that attempts to explain the pleiotropic action of Notch.

A dual laser flow sorter utilizing a CW pumped dye laser.

A dual laser flow sorter has been constructed from an existing single laser system by incorporating a dye laser as the second laser source. The argon ion laser emission was used both as a pump laser and as a source by beam splitting before entrance to the dye laser. The emissions of the dye laser and pump laser beams were recombined and focused with the same optical train used in the single laser system. The imaging on the stream of the flow sorter provided for spatial and thus temporal separation of the exciting beams and subsequent sample emissions.

Selection of viable cells with known DNA content.

Cells doubly stained with Hoechst 33342 (H-33342) for DNA content and fluorescein diacetate for viability can be selected on the basis of both criteria using a single UV laser flow sorter. The selection is made possible due to resonance energy transfer occurring between the H-33342 and fluorescein fluorophores. Both a static fluorescence microscope and a dual laser flow sorter were used to demonstrate that energy transfer occurs in the doubly stained cells and that the sensitized emission in conjunction with the DNA emission can be used to select populations of cells with known DNA content and viability. The results indicate that fluorescein liberated by cellular esterases is freely accessible to the nucleus.

Fluorescence polarization and pulse width analysis of chromosomes by a flow system.

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.