scholarly journals RNA-Seq Atlas—a reference database for gene expression profiling in normal tissue by next-generation sequencing

2012 ◽  
Vol 28 (8) ◽  
pp. 1184-1185 ◽  
Author(s):  
Markus Krupp ◽  
Jens U. Marquardt ◽  
Ugur Sahin ◽  
Peter R. Galle ◽  
John Castle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Normal Tissue ◽  
Reference Database ◽  
Rna Seq ◽  
Next Generation ◽  
Generation Sequencing

243 GENE EXPRESSION PROFILING BY NEXT-GENERATION SEQUENCING OF cDNA SAMPLES FROM INNER CELL MASS AND TROPHECTODERM OF A SINGLE-DAY 12 PORCINE EMBRYO

2010 ◽  
Vol 22 (1) ◽  
pp. 279
Author(s):  
S. C. Isom ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Cdna Fragments ◽  
Generation Sequencing

Traditional microarray approaches to gene expression profiling often require RNA or cDNA amplification when working with extremely small or valuable tissue samples.This process is generally viewed as being undesirable because there is potential for bias to be introduced during amplification. Very recently, the so-called next-generation sequencing technologies were adapted for use in global gene expression profiling. Herein we report our efforts to apply these sequencing technologies to assess relative transcript abundances in pre-implantation-stage porcine embryos, without additional nucleic acid amplification before sequencing. As a proof-of-principle experiment, we have isolated total RNA from the embryonic disc (inner cell mass; ICM) and a small piece of trophectoderm (TE) from a Day 12 in vivo-produced embryo, which were estimated to be composed of 500 to 1000 cells each. The RNA was reverse transcribed using oligo-dT priming followed by second-strand cDNA synthesis. The double-stranded cDNA was then randomly sheared by sonication, and 10 ng of double-stranded cDNA fragments was used for sample preparation before sequencing. Prepared cDNA fragments (at 7 picomolar concentrations) were submitted for sequencing using the Illumina/Solexa platform as recommended. The millions of short (36 bp) reads generated by Illumina sequencing for each sample were then aligned to the swine UniGene database from NCBI, allowing for zero or one mismatches. Relative transcript abundances between cell types were profiled by considering the read counts for a given UniGene member as a percentage of the total number of reads generated for each cell type. It was demonstrated that approximately 11 000 and 9000 UniGene members were represented by a normalized average of 5 or more short reads per lane (0.001% of the total) in the ICM and TE samples, respectively. As expected, pluripotency factors, chromatin remodeling components, and cell-cell communication molecules were overrepresented in the ICM sample as compared with the TE sample. Conversely, epithelial determinants, ion transporters, and components of the steroid biosynthesis pathways were more abundant in the TE sample than in the ICM sample. Relative abundances of representative transcripts in these samples were verified by quantitative RT-PCR. In conclusion, we demonstrate the utility of next-generation sequencing technologies for gene expression profiling using even minute tissue samples and show that such analyses are possible even in species without a sequenced genome.

scholarly journals Genetic approaches to study plant responses to environmental stresses

2018 ◽  
Author(s):  
Khaled Moustafa ◽  
Joanna M. Cross
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Systems Biology ◽  
Massively Parallel Sequencing ◽  
Environmental Stresses ◽  
Plant Responses ◽  
Rna Seq ◽  
Next Generation ◽  
Rnase Protection ◽  
Generation Sequencing

The assessment of gene expression levels is an important step toward elucidating gene functions temporally and spatially. Decades ago, typical studies were focusing on a few genes individually, whereas now researchers are able to examine whole genomes at once. The upgrade of throughput levels aided the introduction of systems biology approaches whereby cell functional networks can be scrutinized in their entireties to unravel potential functional interacting components. The birth of systems biology goes hand-in-hand with huge technological advancements and enables a fairly rapid detection of all transcripts in studied biological samples. Even so, earlier technologies that were restricted to probing single genes or a subset of genes still have their place in research laboratories. The objective here is to highlight key approaches used in gene expression analysis in plant responses to environmental stresses, or, more generally, any other condition of interest. Northern blots, RNase protection assays, and qPCR are described for their targeted detection of one or a few transcripts at a once. Differential display and serial analysis of gene expression represent non-targeted methods to evaluate expression changes of a significant number of gene transcripts. Finally, microarrays and RNA-seq (next-generation sequencing) contribute to the ultimate goal of identifying and quantifying all transcripts in a cell under conditions or stages of study. Recent examples of applications as well as principles, advantages, and drawbacks of each method are contrasted. We also suggest replacing the term "Next-Generation Sequencing (NGS)" with another less confusing synonym such as "RNA-seq", "high throughput sequencing", or "massively parallel sequencing" to avoid confusion with any future sequencing technologies.

scholarly journals Genetic approaches to study plant responses to environmental stresses

2018 ◽  
Author(s):  
Khaled Moustafa
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Systems Biology ◽  
Massively Parallel Sequencing ◽  
Environmental Stresses ◽  
Plant Responses ◽  
Rna Seq ◽  
Next Generation ◽  
Rnase Protection ◽  
Generation Sequencing

The assessment of gene expression levels is an important step toward elucidating gene functions temporally and spatially. Decades ago, typical studies were focusing on a few genes individually, whereas now researchers are able to examine whole genomes at once. The upgrade of throughput levels aided the introduction of systems biology approaches whereby cell functional networks can be scrutinized in their entireties to unravel potential functional interacting components. The birth of systems biology goes hand-in-hand with huge technological advancements and enables a fairly rapid detection of all transcripts in studied biological samples. Even so, earlier technologies that were restricted to probing single genes or a subset of genes still have their place in research laboratories. The objective here is to highlight key approaches used in gene expression analysis in plant responses to environmental stresses, or, more generally, any other condition of interest. Northern blots, RNase protection assays, and qPCR are described for their targeted detection of one or a few transcripts at a once. Differential display and serial analysis of gene expression represent non-targeted methods to evaluate expression changes of a significant number of gene transcripts. Finally, microarrays and RNA-seq (next-generation sequencing) contribute to the ultimate goal of identifying and quantifying all transcripts in a cell under conditions or stages of study. Recent examples of applications as well as principles, advantages, and drawbacks of each method are contrasted. We also suggest replacing the term "Next-Generation Sequencing (NGS)" with another less confusing synonym such as "RNA-seq", "high throughput sequencing", or "massively parallel sequencing" to avoid confusion with any future sequencing technologies.

The Next Generation Sequencing Techniques and Application in Drug Discovery and Development

2019 ◽  
pp. 240-259
Author(s):  
Afzal Hussain
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Expression Profiling ◽  
Massively Parallel Sequencing ◽  
Life Sciences ◽  
Rna Seq ◽  
Next Generation ◽  
Parallel Sequencing ◽  
Short Period ◽  
Generation Sequencing

Next-generation sequencing or massively parallel sequencing describe DNA sequencing, RNA sequencing, or methylation sequencing, which shows its great impact on the life sciences. The recent advances of these parallel sequencing for the generation of huge amounts of data in a very short period of time as well as reducing the computing cost for the same. It plays a major role in the gene expression profiling, chromosome counting, finding out the epigenetic changes, and enabling the future of personalized medicine. Here the authors describe the NGS technologies and its application as well as applying different tools such as TopHat, Bowtie, Cufflinks, Cuffmerge, Cuffdiff for analyzing the high throughput RNA sequencing (RNA-Seq) data.

scholarly journals 5′ end–centered expression profiling using cap-analysis gene expression and next-generation sequencing

2012 ◽  
Vol 7 (3) ◽  
pp. 542-561 ◽  
Author(s):  
Hazuki Takahashi ◽  
Timo Lassmann ◽  
Mitsuyoshi Murata ◽  
Piero Carninci
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Expression Profiling ◽  
Next Generation ◽  
Cap Analysis ◽  
Generation Sequencing
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