Using the transcriptome to annotate the genome

Saha, Saurabh; Sparks, Andrew B.; Rago, Carlo; Akmaev, Viatcheslav R.; Wang, Clarence J.; Vogelstein, Bert; Kinzler, Kenneth W.; Velculescu, Victor E.

doi:10.1038/nbt0502-508

Cited by 582 publications

(424 citation statements)

References 17 publications

Supporting

Mentioning

408

Contrasting

Unclassified

Order By: Relevance

“…This makes SAGE useful as a gene discovery tool (Saha et al, 2002). In our study, approximately 5% of the tags (1918) were present in more than one library, but were unambiguously mapped to genomic regions outside any known genes.…”

Section: Discovery Of New Aging-related Genesmentioning

confidence: 69%

Genes that may modulate longevity in C. elegans in both dauer larvae and long-lived daf-2 adults

Ruzanov

Riddle

Marra

et al. 2007

Experimental Gerontology

View full text Add to dashboard Cite

SummaryWe used Serial Analysis of Gene Expression (SAGE) to compare the global transcription profiles of long-lived mutant daf-2 adults and dauer larvae, aiming to identify aging-related genes based on similarity of expression patterns. Genes that are expressed similarly in both long-lived types potentially define a common life-extending program. Comparison of eight SAGE libraries yielded a set of 120 genes, the expression of which was significantly different in long-lived worms versus normal adults. The gene annotations indicate a strong link between oxidative stress and life span, further supporting the hypothesis that metabolic activity is a major determinant in longevity. The SAGE data show changes in mRNA levels for electron transport chain components, elevated expression of glyoxylate shunt enzymes and significantly reduced expression for components of the TCA cycle in longer-lived nematodes. We propose a model for enhanced longevity through a cytochrome c oxidase-mediated reduction in reactive oxygen species commonly held to be a major contributor to aging.

show abstract

Section: Discovery Of New Aging-related Genesmentioning

confidence: 69%

Genes that may modulate longevity in C. elegans in both dauer larvae and long-lived daf-2 adults

Ruzanov

Riddle

Marra

et al. 2007

Experimental Gerontology

View full text Add to dashboard Cite

show abstract

“…SAGE libraries were sequenced at random using a DNA sequencer (ABI PRISM 377; PE Biosystems, Foster City, CA, USA) and sequence kit (BigDye Terminator Cycle Sequence Kit; PE Biosystems). An NGT library was constructed with the original SAGE protocol (using ten base pair sequence tags), whereas the library for patients with type 2 diabetes was constructed with the long SAGE method (using 17 base pair sequence tags) [16]. Sequenced files were analysed with the SAGE 2000 software (http://www.sagenet.org).…”

Section: Methodsmentioning

confidence: 99%

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes

et al. 2006

View full text Add to dashboard Cite

Aims/hypothesis Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown. Materials and methods We analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT. Results Both gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-α or peroxisome proliferator-activated receptor-γ, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p=0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-γ coactivator-1α or nuclear respiratory factor 1. Conclusions/interpretation Our results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.

show abstract

“…Therefore, the present method may enable RNAi-based forward genetics in which one can identify genes by selection based on absolute phenotypic criteria, such as cell morphology, adhesion, cell death, expression level of a key molecule or other functional indexes. shRNA sequences can serve as reliable tags for gene identification, because the probabilities of 9.1 × 10 -13 and 2.3 × 10 -13 that tags of 20 nt and 21 nt, respectively, with random sequences can be perfectly matched 25 are sufficiently small, considering the size of the mouse or human genome (∼3 × 10 9 nt).…”

Section: Discussionmentioning

confidence: 99%

“…We recovered shRNA constructs by PCR amplification from surviving cells and reconstituted them into retrovirus expression vectors to obtain the selected library. Relative reduction (25) To test whether the ganciclovir selection concentrated efficient shRNA constructs, we infected Jurkat T cells expressing GFP with a mixture of viruses prepared from the reconstituted library. Flow cytometry showed that GFP expression was more attenuated in cells transduced with retroviruses from the ganciclovir-selected library than in cells transduced with retroviruses from the untreated or parental library, indicating that ganciclovir selection concentrated the more efficient shRNA constructs (Fig.…”

Section: Selection Of Efficient Shrna Constructs In Cellsmentioning

confidence: 99%

Enzymatic production of RNAi libraries from cDNAs

et al. 2004

View full text Add to dashboard Cite

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. Highthroughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.Taking advantage of the huge quantity of genome data currently available 1-5 , reverse genetics approaches that can determine the role of each gene by loss-of-function are important in determining gene-function relationships. Gene targeting 6 using homologous recombination is widely used, but its labor-intensive processes preclude its convenient and versatile application. Although antisense oligonucleotides can be more conveniently used, outcomes from this approach are often characterized by toxicity, instability and nonspecific effects 7 .RNAi, a gene suppression phenomenon triggered by doublestranded RNA 8 , is a good alternative 7,9 , because it is usually specific and can be achieved with unprecedented speed in a wide range of organisms. RNAi has been applied to genome-wide reverse genetics in Caenorhabditis elegans 10 . But the initial use of RNAi had been limited to invertebrates because long (>30 nucleotides, nt) double-stranded RNAs elicit interferon responses in higher vertebrates. This problem was overcome by the finding that a short (21-23 nt) double-stranded RNA (siRNA) directs RNAi in mammals without adverse effects 11 . The transient nature of siRNA effects has been overcome by the development of DNA-based vectors by which siRNA or shRNA is expressed intracellularly [12][13][14][15][16][17] .Despite this progress, there are still no general rules for designing siRNA or shRNA constructs with efficient gene-silencing activity; thus, it costs much time and money to identify suitable constructs. We developed a new technology called EPRIL to produce a library of shRNA expression constructs that can be systematically screened. We have also developed a technology for selecting the most efficient shRNA constructs from the library using the thymidine kinase gene. We furthermore showed that EPRIL can be used to produce an RNAi library from a cDNA library. RESULTS Production of an RNAi libraryEPRIL comprises several steps of enzymatic treatments to produce an shRNA expression vector library from cDNAs of interest (Fig. 1a). First, double-stranded DNAs are quasi-randomly fragmented with DNase I (ref. 18). The fragments are...

show abstract

Using the transcriptome to annotate the genome

Cited by 582 publications

References 17 publications

Genes that may modulate longevity in C. elegans in both dauer larvae and long-lived daf-2 adults

Genes that may modulate longevity in C. elegans in both dauer larvae and long-lived daf-2 adults

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes

Enzymatic production of RNAi libraries from cDNAs

Contact Info

Product

Resources

About