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Laser-controlled microdissection of tissues opens a window of new opportunities.

Gene expression analysis using total RNA of bulk tissue usually cannot assign specific messages to particular cell types. Cell-specific RNA expression profiling, though, may be crucial for a better understanding ofthe role of each distinct cell type within a physiological or pathophysiological setting. RNA profiling based on laser-controlled microdissection (LCM) of defined cells of a tissue now provides a useful tool for studying molecular crosstalk between different cell types within a tissue. The LCM technique allows for efficient isolation of single cells with no or very low contamination of surrounding tissue components, simultaneously leaving the intracellular structure and molecules intact. In this review, different issues of the LCM technique and the RNA amplification procedure for microarray analysis are discussed. An exemplary summary of results obtained from gene profiling of epithelial and stromal cells from human prostate tumors is presented, demonstrating the power of LCM-based molecular analysis. Finally, we discuss the potential use of the LCM technique i) to study the transcriptome of distinct cells from formalin-fixed and paraffin-embedded tissues in subcellular RNA profiling and ii) high resolution proteomic and metabolistic studies.

Dissection↗

SAGE profiling and demonstration of differential gene expression along the axial developmental gradient of lignifying xylem in loblolly pine (Pinus taeda).

Wood formation has been studied extensively at the cellular and biochemical levels, but remains poorly understood with respect to gene expression and regulation. As a first step toward identifying genes specifically involved in wood formation and characterizing their roles in determining wood quality, serial analysis of gene expression (SAGE) was used to quantify gene expression in lignifying xylem from a single, 10-year-old loblolly pine (Pinus taeda L.). Two SAGE libraries were generated based on lignifying xylem isolated from either the upper (crown) or lower (base) portions of the trunk. Over 85,000 tags representing a maximum of 27,398 expressed genes were analyzed from the crown wood library, and more than 65,000 tags, representing a maximum of 25,983 expressed genes, were analyzed in the base wood library. Combining these data sets to reflect the sum of genes expressed in lignifying xylem, 150,855 tags were cataloged, representing a maximum of 42,641 different genes. Currently, this study represents the most extensive analysis of its kind in a higher plant and provides a quantitative description of the transcriptome representing the lignifying xylem of a 10-year-old loblolly pine.

DNA, Complementary↗

Complex Genetics and Regulatory Drivers of Hypermobile Ehlers-Danlos Syndrome: Insights from Genome-Wide Association Study Meta-analysis.

BACKGROUND: Hypermobile Ehlers-Danlos syndrome (hEDS) is the most common subtype of EDS, a group of heritable connective tissue disorders. Clinically, hEDS is defined by generalized joint hypermobility and chronic musculoskeletal pain, but its impact extends beyond the musculoskeletal system. Affected individuals frequently experience autonomic, gastrointestinal, immune, and neuropsychiatric involvement, highlighting both the multisystemic nature of the condition and challenges of diagnosis. In contrast to other EDS subtypes with defined genetic causes, the molecular basis of hEDS has remained elusive. METHODS: We conducted a genome-wide association study (GWAS) of hEDS across three case controls studies, including 1,815 cases and 5,008 ancestry-matched controls. Fixed-effects meta-analysis of 6.2 million variants was complemented with LDAK gene-based association testing, transcriptome-wide association studies, and integrative annotation across multiple tissues and cell types including eQTLs, enhancer marks and open chromatin accessibility profiles, supported by luciferase assays on one candidate variant. LD-score genetic correlations were assessed between hEDS and 19 frequently reported comorbid conditions. RESULTS: Two loci reached genome-wide significance, including a regulatory region near the atypical chemokine receptor 3 gene (ACKR3) on chromosome 2. Functional annotation supports ACKR3 risk alleles colocalize with eQTLs in tibial nerve, alter enhancer activity, and generate a de novo AHR transcription factor regulatory site, implicating neuroimmune and pain signaling pathways. Gene-based and transcriptome-wide analyses identified common variants in a locus containing multiple candidates, including SLC39A13, a zinc transporter critical for connective tissue development previously implicated in a rare form of EDS, and PSMC3, a gene involved in central nervous system development. LD-score regression revealed significant genetic correlations between hEDS and joint hypermobility, myalgic encephalomyelitis/chronic fatigue syndrome, fibromyalgia, depression, anxiety, autism spectrum disorder, migraine, and gastrointestinal diseases. CONCLUSIONS: These results establish the first evidence of common variant contributions to hEDS, supporting a complex, multisystem model involving neuroimmune-stromal dysregulation. Our findings add novel indications to hEDS pathogenesis and provide solid foundations for future molecular definition and therapeutic discovery.

Genome-wide association study↗

Gibberellin mobilizes distinct DELLA-dependent transcriptomes to regulate seed germination and floral development in Arabidopsis.

Severe Arabidopsis (Arabidopsis thaliana) gibberellin (GA)-deficient mutant ga1-3 fails to germinate and is impaired in floral organ development. In contrast, the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant confers GA-independent seed germination and floral development. This fact suggests that GA-regulated transcriptomes for seed germination and floral development are DELLA dependent. However, it is currently not known if all GA-regulated genes are GA regulated in a DELLA-dependent fashion and if a similar set of DELLA-regulated genes is mobilized to repress both seed germination and floral development. Here, we compared the global gene expression patterns in the imbibed seeds and unopened flower buds of the ga1-3 mutant with that of the wild type and of the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant. We found that about one-half of total GA-regulated genes are apparently regulated in a DELLA-dependent fashion, suggesting that there might be a DELLA-independent or -partially-dependent component of GA-dependent gene regulation. A cross-comparison based on gene identity revealed that the GA-regulated DELLA-dependent transcriptomes in the imbibed seeds and flower buds are distinct from each other. Detailed ontology analysis showed that, on one hand, DELLAs differentially regulate the expression of different individual members of a gene family to run similar biochemical pathways in seeds and flower. Meanwhile, DELLAs control many functionally different genes to run specific pathways in seeds or flower buds to mark the two different developmental processes. Our data shown here not only confirm many previous reports but also single out some novel aspects of DELLA functions that are instructive to our future research.

Amino Acid Sequence↗

The structured mRNA element 45ABC mediates auto- and cross-regulation of RBP45 genes via alternative splicing.

Alternative splicing (AS) is a common gene regulatory mechanism involving distinct interactions between trans-acting factors and cis-regulatory elements on the precursor messenger RNA (pre-mRNA). In this study, we have functionally characterized the structured motif 45ABC, which is located in the pre-mRNAs of RNA-binding protein (RBP) 45 genes in many plant species. Our data revealed that this element mediates a negative auto- and cross-regulatory feedback loop via AS of the three 45ABC-containing RBP45 genes in Arabidopsis thaliana. We identified a G-rich stretch within the first stem as a potential RBP45 binding site and observed increased RBP45-dependent AS upon structural weakening of this pairing element. The second stem includes the alternative 5' splice site being activated in the presence of RBP45. Based on the known interaction between RBP45 homologs and U1 snRNP components required for 5' splice site recognition, we propose that RBP45 recruitment to stem I of 45ABC may induce usage of the alternative 5' splice site in stem II. The resulting splicing variant is unproductive, thereby diminishing RBP45 expression. Analysing the splicing-regulatory impact of the three At-RBP45 genes in auto- and cross-regulation and a transcriptome-wide manner revealed unequal genetic redundancy with a major role of RBP45B. Furthermore, phenotypical analysis of single- and higher-order rbp45 mutants pointed at these genes' functions in controlling primary root growth and flowering time. Taken together, we demonstrated that both sequence and structural features of 45ABC are critical for proper splicing control, balancing RBP45 expression and functions in plants via a conserved mRNA motif.

Alternative Splicing↗

Impaired natural killer cell maturation in lung adenocarcinoma driven by FABP4 and SPON2 downregulation through disrupted lipid metabolism.

BACKGROUND: Although natural killer (NK) cells play a crucial role in antitumor immunity, the metabolic changes driving their dysfunction in lung adenocarcinoma remain poorly understood. This study investigates how these metabolic modifications impact NK cell function within the lung adenocarcinoma microenvironment. METHODS: A total of 13 pairs of lung adenocarcinoma samples were obtained from The Cancer Genome Atlas. Differential gene expression, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and single-cell metabolic quantification analyses were used to characterize the transcriptomic, pathway, and metabolic signatures of NK cells. The developmental trajectory was reconstructed via pseudotime analysis. The fatty acid-binding protein 4 (FABP4) and spondin2 (SPON2) expression was examined using immunofluorescence (IF) and immunohistochemistry (IHC) in patients with lung adenocarcinoma. In NK cells with FABP4 downregulation, FABP4 function was analyzed using antibody-independent cell-mediated cytotoxicity assays, flow cytometry (FCM), and liquid chromatography-mass spectrometry. RESULTS: The number of NK cells was significantly decreased in the lung adenocarcinoma microenvironment. FABP4 and SPON2 expression was significantly lower in NK cells within tumor tissues than in the adjacent tissues. FABP4 expression was significantly lower in tumor tissues than in the adjacent tissues, whereas no significant difference in SPON2 expression was observed. The cytotoxic function of NK cells with decreased FABP4 levels was impaired. Non-targeted lipid metabolism analysis indicated that differentially expressed lipids in NK cells with low FABP4 levels were functionally enriched in the glycerophospholipid metabolism pathway compared to those in normal NK cells. CONCLUSIONS: The study findings present new evidence showing that low FABP4 and SPON2 gene expression may impair NK cell maturity by affecting lipid metabolism in lung adenocarcinoma. These results provide a new perspective on restoring immune function in patients with lung cancer.

FABP4↗

Identification of genes selectively regulated by IFNs in endothelial cells.

IFNs are highly pleiotropic cytokines also endowed with marked antiangiogenic activity. In this study, the mRNA expression profiles of endothelial cells (EC) exposed in vitro to IFN-alpha, IFN-beta, or IFN-gamma were determined. We found that in HUVEC as well as in other EC types 175 genes were up-regulated (>2-fold increase) by IFNs, including genes involved in the host response to RNA viruses, inflammation, and apoptosis. Interestingly, 41 genes showed a >5-fold higher induction by IFN-alpha in EC compared with human fibroblasts; among them, the gene encoding the angiostatic chemokine CXCL11 was selectively induced by IFN-alpha in EC along with other genes associated with angiogenesis regulation, including CXCL10, TRAIL, and guanylate-binding protein 1. These transcriptional changes were confirmed and extended by quantitative PCR analysis and ELISA; whereas IFN-alpha and IFN-beta exerted virtually identical effects on transcriptome modulation, a differential gene regulation by type I and type II IFN emerged, especially as far as quantitative aspects were concerned. In vivo, IFN-alpha-producing tumors overexpressed murine CXCL10 and CXCL11, guanylate-binding protein 1, and TRAIL, with evidence of CXCL11 production by tumor-associated EC. Overall, these findings improve our understanding of the antiangiogenic effects of IFNs by showing that these cytokines trigger an antiangiogenic transcriptional program in EC. Moreover, we suggest that quantitative differences in the magnitude of the transcriptional activation of IFN-responsive genes could form the basis for cell-specific transcriptional signatures.

Animals↗

STN1 upregulation promotes PARPi resistance in BRCA2-deficient cancer cells via replication fork protection and suppression of ssDNA gap formation.

PARPi are effective therapy for BRCA1/2 mutant cancers, yet recurrent PARPi resistance frequently develops. The underlying mechanism of PARPi resistance remains largely unresolved. Here, we identify STN1, a component of the CTC1/STN1/TEN1 (CST) complex, as a modulator of PARPi resistance in BRCA2-deficient cells. RNA-seq analysis of PARPi-resistant cancer cells from BRCA2-mutated backgrounds shows largely distinct transcriptomic profiles with limited overlap, suggesting multiple routes to resistance. Notably, STN1 is consistently upregulated in resistant cells. We observe that overexpression of STN1 enhances Olaparib resistance in multiple BRCA2-deficient cell lines and alleviates DNA damage under replication stress. Mechanistically, we find that STN1 overexpression increases RAD51 loading to stalled replication forks while restricting MRE11 recruitment in BRCA2-deficient cells, thereby protecting stalled forks from nascent-strand degradation. Furthermore, STN1 overexpression rescues the accumulation of ssDNA gaps, a major determinant of PARPi sensitivity in BRCA2-deficient cells. Taken together, these findings suggest that elevated STN1 levels can partially compensate for BRCA2 loss by stabilizing stalled replication forks and limiting ssDNA gap accumulation. Our study uncovers a STN1-dependent pathway of replication stress tolerance that promotes PARPi resistance independently of homologous recombination restoration, highlighting STN1 as a potential biomarker and mechanistic contributor to therapeutic resistance in BRCA2-mutated cancers.

PARPi resistance↗

DNA microarrays in pediatric cancer.

Childhood cancer, like all cancer, is at heart a genetic disease. Consequently, fundamental understanding of the oncogenic process is likely to be beneficially addressed by genetic methodology. Current methods have largely focused on single-gene defects, like chimeric genes, which are present in many sarcomas and leukemias. Real understanding is more likely to derive from a genome-wide analysis of these malignancies. Recent technologic advances have made it possible to simultaneously assess the entire expressed gene profile, or transcriptome, of a given cancer. Foremost among these methods is gene expression profiling using DNA microarrays. Two basic approaches predominate: spotted arrays and photolithography arrays. Regardless of the method, the resulting information can be used to create disease profiles, but only if appropriate bioinformatic solutions are employed. Common analytic approaches include two-way expression comparisons, or scatter analyses; outlier gene analysis, to identify significantly dysregulated genes; dendrogram analyses, as pioneered by Eisen; cluster analyses to identify diagnostic or biologic groups; and various forms of functional analyses to identify relevant genes and biologic pathways. Studies of both adult and pediatric cancer have demonstrated the feasibility of such analyses to identify both diagnostic and prognostic groups of tumors. Acute childhood leukemias have been grouped into myelogenous and lymphoid, and even B- and T-cell subsets. Breast cancer prognostic groups have been identified on the basis of a small subset of expressed genes. In addition, preliminary data on childhood sarcomas appear to identify both diagnostic and prognostic subsets. Specifically, embryonal rhabdomyosarcoma could be distinguished from alveolar rhabdomyosarcoma, and even morphologically mixed embryonal and alveolar rhabdomyosarcoma showed similar gene expression profiles in both histologies. Further, collaborative studies using clustering analyses appear to identify prognostic groups of diverse sarcomas. Larger institutional and cooperative group studies are currently underway to validate these preliminary findings.

Animals↗

Single-cell glycome and transcriptome profiling enabled by a library of anti-glycan antibodies.

Glycans play critical roles in cellular processes and clinical applications, but they remain difficult to study due to a shortage of well-characterized anti-glycan reagents and high-throughput technologies for glycome profiling, especially ones capable of single-cell resolution. To meet these needs, we generated a database of 650 anti-glycan antibody sequences, recombinantly expressed a library of 154 antibodies, and extensively characterized their binding properties using glycan microarrays. In addition to providing valuable information and resources for the field, the sequence database and microarray data also enabled development of "Glycomic-seq" (Glycome profiling via multiplexed immunoglobulins combined with sequencing), a DNA-barcoded anti-glycan antibody platform that enables high-throughput, single-cell profiling of both RNA and cell-surface glycan expression. Using Glycomic-seq, we profiled two isogenic colorectal cancer cell lines. The results revealed various glycans associated with cancer stem cells and metastasis, demonstrating the power of integrating glycomic information with multi-omic efforts to discover biomarkers and therapeutic targets.

Polysaccharides↗

Genomewide nonadditive gene regulation in Arabidopsis allotetraploids.

Polyploidy has occurred throughout the evolutionary history of all eukaryotes and is extremely common in plants. Reunification of the evolutionarily divergent genomes in allopolyploids creates regulatory incompatibilities that must be reconciled. Here we report genomewide gene expression analysis of Arabidopsis synthetic allotetraploids, using spotted 70-mer oligo-gene microarrays. We detected >15% transcriptome divergence between the progenitors, and 2105 and 1818 genes were highly expressed in Arabidopsis thaliana and A. arenosa, respectively. Approximately 5.2% (1362) and 5.6% (1469) genes displayed expression divergence from the midparent value (MPV) in two independently derived synthetic allotetraploids, suggesting nonadditive gene regulation following interspecific hybridization. Remarkably, the majority of nonadditively expressed genes in the allotetraploids also display expression changes between the parents, indicating that transcriptome divergence is reconciled during allopolyploid formation. Moreover, >65% of the nonadditively expressed genes in the allotetraploids are repressed, and >94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa, consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica. The nonadditive gene regulation is involved in various biological pathways, and the changes in gene expression are developmentally regulated. In contrast to the small effects of genome doubling on gene regulation in autotetraploids, the combination of two divergent genomes in allotetraploids by interspecific hybridization induces genomewide nonadditive gene regulation, providing a molecular basis for de novo variation and allopolyploid evolution.

Arabidopsis↗

Identification of a new Schistosoma mansoni membrane-bound protein through bioinformatic analysis.

Progress in schistosome genome research has enabled investigators to move rapidly from genome sequences to vaccine development. Proteins bound to the surface of parasites are potential vaccine candidates, or they can be used for diagnosis. We analyzed 4342 proteins deduced from the Schistosoma mansoni transcriptome with bioinformatic computer programs. Thirty-four proteins had membrane-bound motifs. Within this group, we selected the Sm29 protein to be further characterized by in silico analysis. Sm29 was found to have a signal peptide made up of 26 amino acids, with a cleavage site between Ser26 and Val27. The glycosylation site search revealed three threonines (39, 132 and 133) with high probability of O-glycosylation and two asparagines (58 and 115) with high probability of N-glycosylation. Only one transmembrane helix was found in the C-terminal region of the protein from Leu169 to Lis191. The search for similarities and conserved motifs show that Sm29 is a protein with high identity to proteins present in S. japonicum (53, 52, 49, and 37% of identity) and it possesses disulfide-rich conserved domains. Apparently, Sm29 is a membrane bound protein, and it may be an important molecule in host-parasite interactions.

Amino Acid Sequence↗

PoweREST: Statistical Power Estimation for Spatial Transcriptomics Experiments to Detect Differentially Expressed Genes Between Two Conditions.

Recent advancements in Spatial Transcriptomics (ST) have significantly enhanced biological research in various domains. However, the high cost of current ST data generation techniques restricts its application in large-scale population studies. Consequently, there is a pressing need to maximize the use of available resources to achieve robust statistical power. One fundamental question in ST analysis is to detect differentially expressed genes (DEGs) among different conditions using ST data. Such DEG analysis is often performed but the associated power calculation is rarely discussed in the literature. To address this gap, we introduce, PoweREST (https://github.com/lanshui98/PoweREST), a power estimation tool designed to support power calculation of DEG detection with 10X Genomics Visium data. PoweREST enables power estimation both before any ST experiments or after preliminary data are collected, making it suitable for a wide variety of power analyses in ST studies. We also provide a user-friendly, program-free web application (https://lanshui.shinyapps.io/PoweREST/), allowing users to interactively calculate and visualize the study power along with relevant the parameters.

Differentially expressed genes↗

Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE.

PURPOSE: To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). METHODS: Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. RESULTS: Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. CONCLUSIONS: The EyeSAGE database, combining three different gene-profiling platforms including the authors' multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.

Adult↗

Comparative analysis of 5,211 leaf ESTs of wild rice (Oryza minuta).

The expressed sequence tags (ESTs) presented in this report are the first transcriptomes of wild rice. A cDNA library was constructed from 4-week-old leaf samples of greenhouse-grown Oryza minuta. The 5,211 cDNA clones of O. minuta represent 3,401 unique sequences, consisting of 2,787 singletons and 614 assembled sequences. Database comparisons of the cDNAs in GenBank's non-redundant databases using BLAST revealed that 4,957 of the 5,211 cDNAs (95.1%) showed a high degree of sequence homology to genes from other organisms. Most of the transcripts identified were genes related to metabolism, energy, protein biosynthesis and subcellular localization. The metabolism and energy categories of the O. minuta ESTs showed a considerably higher gene expression level than those of O. sativa ESTs. These data and genes can be utilized in rice breeding.

Arabidopsis↗

DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic stem cells. Despite the high incidence of MDS in the elderly, effective treatment of individuals in its advanced stages is problematic. DNA microarray analysis is a potentially informative approach to the development of new treatments for MDS. However, a simple comparison of 'transcriptomes' of bone marrow mononuclear cells among individuals at distinct stages of MDS would result in the identification of genes whose expression differences only reflect differences in the proportion of MDS blasts within bone marrow. Such a 'population shift' effect has now been avoided by purification of haematopoietic stem-like cells that are positive for the cell surface marker AC133 from the bone marrow of healthy volunteers and 30 patients at various stages of MDS. Microarray analysis with the AC133+ cells from these individuals resulted in the identification of sets of genes with expression that was specific to either indolent or advanced stages of MDS. The former group of genes included that for PIASy, which catalyses protein modification with the ubiquitin-like molecule SUMO. Induction of PIASy expression in a mouse myeloid cell line induced apoptosis. A loss of PIASy expression may therefore contribute directly to the growth of MDS blasts and stage progression.

Acute Disease↗

Salivary transcriptome diagnostics for oral cancer detection.

PURPOSE: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease. EXPERIMENTAL DESIGN: Unstimulated saliva was collected from patients (n = 32) with primary T1/T2 OSCC and normal subjects (n = 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models. RESULTS: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05). Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n = 32) and controls (n = 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls. CONCLUSIONS: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

Biomarkers, Tumor↗

Statistical issues in microarray data analysis.

Microarrays provide the ability to quantitatively measure the abundance of specific RNA transcripts through sample hybridization to a solid-state grid of oligonucleotides or amplicons. The prospect of measuring the entire transcriptome is extremely alluring, but as with any experiment, it should be met with caution and great consideration. The level of confidence we can assign to the results depends on the skill at which the experiment is conducted, the quality of the experimental design and subsequent analysis, and, most important, the power in the study. Any microarray experiment consists of several components: (1) carrying out an appropriately designed (replicated) plant experiment; (2) array processing, which includes several steps of data acquisition and normalization; and (3) analysis of expression data to identify differentially expressed genes and overall patterns of expression. Numerous software packages are available to assist in performing these steps and it is not our intent to provide a software users manual or a statistical review. It is our intent to provide a brief user's explanation of these various components and present the commonly used methods.

Algorithms↗