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Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses. [ABSTRACT FROM AUTHOR]

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFα expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992–hnRNPL complex that regulated transcription of the TNFα gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFα expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFα and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFα expression and may play important roles in the innate immune response and inflammatory diseases in humans. [ABSTRACT FROM AUTHOR]

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of l|i germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChlP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. [ABSTRACT FROM AUTHOR]

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity. [ABSTRACT FROM AUTHOR]

Background: Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. Results: From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r² values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r² values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2. Conclusions: Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data. [ABSTRACT FROM AUTHOR]

The usage of >100 functional murine Ig heavy chain VH genes, when rearranged to DHJH genes, generates a diverse antibody repertoire. The VH locus encompasses 2.5 Mb, and rearrangement of VH genes in the DH-distal half of the locus are controlled very differently from the VH genes in the proximal end of the locus. The rearrangement of distal but not proximal VH genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5. or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal VH rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal VH segments and the repressive histone modification H3K27me3 is exclusively present on proximal VH segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal VH genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the VH locus by differentially modulating two reciprocal histone modifications during B lymphocyte development. [ABSTRACT FROM AUTHOR]

We have developed methods for using DNA array technology to probe the entire transcriptome to determine the RNA-binding specificity of ligands. Two methods were investigated. In the first method, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal was compared with a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect that the results will lead to the discovery of new aminoglycosidebinding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic. [ABSTRACT FROM AUTHOR]

CNS abnormalities can be detected during chronic human immunodeficiency virus (HIV) infection, before the development of opportunistic infections or other sequelae of immunodeficiency. However, although end-stage dementia caused by HIV has been linked to the presence of infected and activated macrophages and microglia in the brain, the nature of the changes resulting in the motor and cognitive disorders in the chronic stage is unknown. Using simian immunodeficiency virus-infected rhesus monkeys, we sought the molecular basis for CNS dysfunction. In the chronic stable stage, nearly 2 years after infection, all animals had verified CNS functional abnormalities. Both virus and infiltrating lymphocytes (CD8+ T-cells) were found in the brain. Molecular analysis revealed that the expression of several immune response genes was increased, including CCL5, which has pleiotropic effects on neurons as well as immune cells. CCL5 was significantly upregulated throughout the course of infection, and in the chronic phase was present in the infiltrating lymphocytes. We have identified an altered state of the CNS at an important stage of the viral-host interaction, likely arising to protect against the virus but in the long term leading to damaging processes. [ABSTRACT FROM AUTHOR]

purpose. Glycoconjugates regulate a variety of biological events in mucosal surfaces, such as differentiation of postmitotic epithelial cells and maintenance of the wet-surfaced phenotype. This study aimed to identify the repertoire of genes (glycogenes) involved in biosynthesis of glycoconjugates in conjunctiva of normal subjects and patients with dry eye. methods. RNA from conjunctival impression cytology samples was amplified and hybridized to a custom-designed glycogene microarray. Intensity data were converted to expression values and analyzed by ANOVA. Microarray data for selected Notch glycogenes were confirmed by quantitative real-time PCR. Notch receptors and ligands were immunolocalized on conjunctival biopsies by confocal microscopy. results. By microarray, 424 glycogenes were identified in normal conjunctival epithelium; galectins, glycosyltransferases, mucins, Notch signaling molecules, and proteoglycans were among the most highly expressed. In dry eye, 46 glycogenes were significantly downregulated, including five members of the Notch signaling pathway (Notch1, Notch 2, Notch 3, Jagged1, Delta1), four Wnt signaling molecules (Wnt4, -5A, Frizzled6, -7), and three heparan sulfate glycotransferases (HS2ST1, HS3ST6, EXTL2). Only interferon-induced transmembrane protein 1 was upregulated. By real-time PCR, expression ratios of Notch1, Notch 3, and Jagged1 in dry eye were 0.43, 0.56, and 0.50, respectively, compared to controls (P < 0.05). Notch1, Notch3, and Jagged1 were immunolocalized throughout the conjunctival epithelium, whereas Notch2 and Delta1 were distributed apically. conclusions. This study revealed the differential glycogene expression profiles in normal subjects and patients with dry eye. Downregulation of Notch signaling in dry eye may result in abnormal differentiation of the conjunctival epithelium and have implications in the pathogenesis of the disease.

Abstract: Background: Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. Methods: The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. Results and conclusions: One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewisx motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewisx structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewisx, as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. General Significance: These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewisx motif plays a functional role in neurite outgrowth from human neurons. [Copyright &y& Elsevier]

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this chapter, we describe a method for making Illumina-compatible sequencing libraries from RNA. This protocol can be used for standard RNAseq analysis for detecting differentially expressed genes. In addition, this protocol is ideally suited for adapting to RIPseq, 5'-RACE, RNA structural probing, nascent RNA sequencing, and other protocols where polymerase termination sites need to be profiled. The utilization of solid-phase bead chemistries facilitates simple workflow and efficient library yields.

Many methods exist for examining CpG DNA methylation. However, many of these are qualitative, laborious to apply to a large number of genes simultaneously, or are not easy to target to specific regions of interest. Microdroplet PCR-based bisulfite sequencing allows for quantitative single base resolution analysis of investigator selected regions of interest. Following bisulfite conversion of genomic DNA, targeted microdroplet PCR is conducted with custom primer libraries. Samples are then fragmented, concatenated, and sequenced by high-throughput sequencing. The most recent technology allows for this method to be conducted with as little as 250 ng of bisulfite-converted DNA. The primary advantage of this method is the ability to hand-select the targeted regions covered by up to 10,000 amplicons of 500-600 bp. Moreover, the nature of microdroplet PCR virtually eliminates PCR bias and allows for the amplification of all targets simultaneously in a single tube.

With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.

Transcriptome analysis or global gene expression profiling is a powerful tool for discovery as well as -understanding biological mechanisms in health and disease. We present in this chapter a description of methods used to isolate mRNA from cells and tissues that has been optimized for preservation of RNA quality using clinical materials and implemented successfully in several large, multicenter studies by the authors. In addition, two methods, gene expression microarrays and RNAseq, are described for mRNA profiling of cells and tissues from clinical or laboratory sources.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1(-/-) pro-B cells are greatly impaired in distal V(H) gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V(H) genes close to the DJ(H) rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V(H) genes to undergo efficient rearrangement.

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity., Competing Interests: The authors have declared that no competing interests exist.

Glycans are vital elements of living organisms and are involved in recognition, communication, cell growth and development, motility, and other significant processes. The interactions of glycans with the proteins that bind them provide valuable information about protein interaction and specificity. By printing glycans on microarrays, investigators are able to effectively determine the binding specificity of certain proteins with an extremely efficient and precise result. Such chips are performed by standard robotic microarray printing. Incubating the slides with various GBP-containing substances not only reveals clear receptor preferences of the proteins, but also detects minute differences in structure specificity.

In this chapter, we describe numerous methods to extract RNA, DNA, and protein from tissue, represented by kidney transplant biopsies, and from peripheral blood cells collected at various clinical sites. Gene expression profiling and SNP-based genome-wide association studies are done using various microarray platforms. In addition, protocols that enable simultaneous protein purification from these clinical samples, enable additional strategies for understanding of the molecular processes involved in organ transplantation, immunosuppressive drug regimens, and the elements determining allograft success and failure. Successfully establishing a multicenter clinical study was essential to meet our objectives for subject enrollment and transplant outcomes. This chapter focuses on our experience setting up and coordinating clinical sample collection from multiple transplant centers for the purpose of microarray analysis.

As the field of glycobiology grows, important roles for glycolipids and glycoproteins in neurological disorders are being increasingly appreciated. However, few studies have explored the involvement of these molecules in the pathology of psychiatric illnesses. We investigated molecular differences related to glycobiology in subjects with schizophrenia by analyzing gene expression profiles using a focused glycogene chip, a custom-designed oligonucleotide array containing genes encoding proteins related to glycobiology, including glycosyltransferases, carbohydrate-binding proteins, proteoglycans, and adhesion molecules. We measured expression profiles in prefrontal cortical (BA46) samples from schizophrenic subjects and matched controls. We find differential expression of genes particularly related to glycosphingolipid/sphingolipid metabolism and N- and O-linked glycan biosynthesis in subjects with schizophrenia. Expression decreases of seven genes associated with these pathways, UGT8, SGPP1, GALC, B4GALT6, SPTLC2, ASAH1, and GAL3ST1, were validated by quantitative PCR in schizophrenic subjects with short-term illness. Only one of these genes, SPTLC2, showed differential expression in chronic schizophrenic subjects, although an increase in expression was observed. Covariate analysis showed that the expression of five of these genes was significantly positively correlated with age in schizophrenic, but not control, subjects. These changing patterns of expression could represent an adaptive response to pathology with disease progression or a compensatory effect of antipsychotic medication, although no significant correlations between gene expression levels and drug doses were observed. Disruption of sphingolipid metabolism early in illness could result in widespread downstream effects encompassing diverse pathological deficits already described in schizophrenia, especially those involving myelination and oligodendrocyte function; hence, this system may represent an important link in schizophrenia pathology., (2008 Wiley-Liss, Inc.)

Helicobacter pylori infects over half the population worldwide and is a leading cause of chronic gastritis and gastric cancer. However, the mechanism by which this organism induces inflammation and carcinogenesis is not fully understood. In the present study we used insulin-gastrin (INS-GAS) transgenic mice that fully develop gastric adenocarcinoma after infection of H. pylori-related Helicobacter felis. Histological examination revealed that more than half of those mice developed invasive adenocarcinoma after 8 months of infection. These carcinomas were stained by NCC-ST-439 and HECA-452 that recognize 6-sulfated and non-sulfated sialyl Lewis X. Lymphocytic infiltration predominantly to submucosa was observed in most H. felis-infected mice, and this was associated with the formation of peripheral lymph node addressin (PNAd) on high endothelial venule (HEV)-like vessels detected by MECA-79. Time-course analysis of gene expression by using gene microarray revealed upregulation of several inflammation-associated genes including chemokines, adhesion molecules, surfactant protein D (SP-D), and CD74 in the infected stomach. Immunohistochemical analysis demonstrated that SP-D is expressed in hyperplasia and adenocarcinoma whereas CD74 is expressed in adenocarcinoma in situ and invasive carcinoma. These results as a whole indicate that H. felis induces HEV-like vessels and inflammation-associated chemokines and chemokine receptors, followed by adenocarcinoma formation.

We have developed methods for using DNA array technology to probe the entire transcriptome to determine the RNA-binding specificity of ligands. Two methods were investigated. In the first method, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal was compared with a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect that the results will lead to the discovery of new aminoglycoside-binding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.

Gene expression analysis has become a standard tool for studies of biological models of disease processes, growth, and development and has been used increasingly in the field of clinical research. The completion of the human genome sequence is now providing the glycomics research community with the tools to develop a synergism with related fields such as MS glycan profiling and proteomics. The use of microarray technology to measure changes in gene expression is a useful approach for the study of the expression profiles of glycosyltransferases, carbohydrate binding proteins, and related enzymes. The availability of standardized microarray platforms, high-quality reagents for sample preparation and hybridization, and image and statistical analysis tools has contributed to recent advances in the application of this technology to glycobiology. Most microarray experiments today are done within specialized core facilities equipped with instrumentation, software, and personnel with expertise in their application. The focus of this chapter is on aspects of microarray analysis common to all platforms and within the realm of the glycobiologist: consideration of experimental design issues and methods for the preparation of RNA samples from a variety of tissue sources suitable for microarray analysis.