• RNA sequencing
• analyses of NGS data
• gene expression
• mRNA transcript variants
• spatial transcriptomics
• verification of NGS data

• Humans
• RNA, Messenger/genetics
• Animals
• Sequence Analysis, RNA/methods
• Genetic Variation
• Alternative Splicing

• Clustered regularly interspaced short palindromic repeat
• DNA sequencing
• Gene based therapy
• Gene sequencing
• Genome editing
• Genomic medicine
• Genomic tools
• Medical genetics
• RNA 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.

• DNA arrays
• NGS
• environmental stress
• gene expression profiling
• gene expression study
• microarrays
• next generation sequencing
• plant adaptation
• plant stress response
• plant stress tolerance

• glucocorticoids
• gene modulation
• kinases
• in vivo treatment
• t cells
• thymocytes
• apoptosis
• knock-out mice

• Adjuvants, Immunologic/pharmacology
• Animals
• Base Sequence
• Down-Regulation/drug effects
• Gene Expression Regulation
• Glucocorticoids/pharmacology
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics
• Mice
• Mice, Inbred BALB C
• Mice, Inbred C3H
• Mice, Knockout
• Molecular Sequence Data
• Polymerase Chain Reaction
• Protein Kinases/genetics
• Protein-Tyrosine Kinases/genetics
• Receptor Protein-Tyrosine Kinases/genetics
• Receptors, Antigen, T-Cell/genetics
• Transcription, Genetic
• Up-Regulation/drug effects

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.