Thomas D. Schmittgen

Bio: Thomas D. Schmittgen is an academic researcher from University of Florida. The author has contributed to research in topics: microRNA & Gene expression. The author has an hindex of 48, co-authored 111 publications receiving 150512 citations. Previous affiliations of Thomas D. Schmittgen include University of Southern California & University of Oklahoma.

HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts

Abstract: Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genomewide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts’ (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category. Cell Death and Differentiation advance online publication, 13 September 2013; doi:10.1038/cdd.2013.119

High-Throughput Real-Time PCR

Abstract: Real-time PCR is presently the gold standard of gene expression quantification. Configuration of real-time PCR instruments with 384-well reaction blocks, enables the instrument to be used essentially as a low-density array. While PCR will never rival the throughput of microchip arrays, in situations where one is interested in assaying several hundreds of genes, high throughput, real-time PCR is an excellent alternative to microchip arrays. By combining SYBR green detection and 5 microL reaction volume, the associated costs of high-throughput real-time PCR are comparable to microarrays. Described here is a complete protocol to perform real-time PCR in a 384-well configuration. Examples are provided to access numerous PCR primer sequences that may be used for high-throughput real-time PCR. Methods of analysis are described to present real-time PCR data as heat maps and clustered similar to the presentation of cDNA microarray data. An example is provided to profile the expression of over 200 microRNA precursors using high-throughput real-time PCR.

Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer

Abstract: Multiple recent studies have found aberrant expression profiles of microRNAome in human cancers. While several target genes have been experimentally identified for some microRNAs in various tumors, the global pattern of cellular functions and pathways affected by co-expressed microRNAs in cancer remains elusive. The goal of this study was to develop a computational approach to global analysis of the major biological processes and signaling pathways that are most likely to be affected collectively by co-expressed microRNAs in cancer cells. We report results of computational analysis of five datasets of aberrantly expressed microRNAs in five human cancers published by the authors (pancreatic cancer) and others (breast cancer, colon cancer, lung cancer and lymphoma). Using the combinatorial target prediction algorithm miRgate and a two-step data reduction procedure we have determined Gene Ontology categories as well as biological functions, disease categories, toxicological categories and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in specific cancers. Our global analysis of predicted miRNA targets suggests that co-expressed miRNAs collectively provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways known to be affected in a particular cancer. Such systems biology based approach provides new avenues for biological interpretation of miRNA profiling data and generation of experimentally testable hypotheses regarding collective regulatory functions of miRNA in cancer.

Dietary apigenin reduces LPS-induced expression of miR-155 restoring immune balance during inflammation

Abstract: Scope High incidence of inflammatory diseases afflicts the increasing aging-population infringing a great health burden. Dietary flavonoids, including the flavone apigenin, are emerging as important anti-inflammatory nutraceuticals due to their health benefits, lack of adverse effects and reduced costs. MicroRNAs (miRs) play a central role in inflammation by regulating gene expression, yet how dietary ingredients affect miRs is poorly understood. The aim of this study was to identify miRs involved in the anti-inflammatory activity of apigenin and apigenin-rich diets and determine their immune regulatory mechanisms in macrophages and in vivo. Methods and results A high-throughput quantitative reverse transcriptase PCR screen of 312 miRs in macrophages revealed that apigenin reduced LPS-induced miR-155 expression. Analyses of miR-155 precursor and primary transcript indicated that apigenin regulated miR-155 transcriptionally. Apigenin-reduced expression of miR-155 led to the increase of anti-inflammatory regulators forkhead box O3a and smooth-muscle-actin and MAD-related protein 2 in LPS-treated macrophages. In vivo, apigenin or a celery-based apigenin-rich diet reduced LPS-induced expression of miR-155 and decreased tumor necrosis factor α in lungs from LPS-treated mice. Conclusion These results demonstrate that apigenin and apigenin-rich diets exert effective anti-inflammatory activity in vivo by reducing LPS-induced expression of miR-155, thereby restoring immune balance.

Analyzing real-time PCR data by the comparative C(T) method.

Abstract: Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

Abstract: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

MicroRNA signatures in human cancers

Abstract: MicroRNA (miRNA ) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.