Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
Briefly, SAGE experiments proceed as follows:
The mRNA of an input sample (e.g. a tumour) is isolated and a reverse transcriptase and biotinylated primers are used to synthesize cDNA from mRNA.
The cDNA is bound to Streptavidin beads via interaction with the biotin attached to the primers, and is then cleaved using a restriction endonuclease called an anchoring enzyme (AE). The location of the cleavage site and thus the length of the remaining cDNA bound to the bead will vary for each individual cDNA (mRNA).
The cleaved cDNA downstream from the cleavage site is then discarded, and the remaining immobile cDNA fragments upstream from cleavage sites are divided in half and exposed to one of two adaptor oligonucleotides (A or B) containing several components in the following order upstream from the attachment site: 1) Sticky ends with the AE cut site to allow for attachment to cleaved cDNA; 2) A recognition site for a restriction endonuclease known as the tagging enzyme (TE), which cuts about 15 nucleotides downstream of its recognition site (within the original cDNA/mRNA sequence); 3) A short primer sequence unique to either adaptor A or B, which will later be used for further amplification via PCR.
After adaptor ligation, cDNA are cleaved using TE to remove them from the beads, leaving only a short "tag" of about 11 nucleotides of original cDNA (15 nucleotides minus the 4 corresponding to the AE recognition site).
The cleaved cDNA tags are then repaired with DNA polymerase to produce blunt end cDNA fragments.
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Cultivated animal cells are important hosts for the production of recombinant proteins for biochemical and structural studies and for use as therapeutics. The course will provide an overview of the me
This course covers various data analysis approaches associated with applications of DNA sequencing technologies, from genome sequencing to quantifying gene expression, transcription factor binding and
In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between cells that are actively dividing, or show how the cells react to a particular treatment. Many experiments of this sort measure an entire genome simultaneously, that is, every gene present in a particular cell. Several transcriptomics technologies can be used to generate the necessary data to analyse.
Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell.
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The term transcriptome is a portmanteau of the words transcript and genome; it is associated with the process of transcript production during the biological process of transcription. The early stages of transcriptome annotations began with cDNA libraries published in the 1980s.
This course will provide the fundamental knowledge in neuroscience required to
understand how the brain is organised and how function at multiple scales is
integrated to give rise to cognition and beh
This course will provide the fundamental knowledge in neuroscience required to
understand how the brain is organised and how function at multiple scales is
integrated to give rise to cognition and beh
This course will provide the fundamental knowledge in neuroscience required to
understand how the brain is organised and how function at multiple scales is
integrated to give rise to cognition and beh
Cis-genetic effects are key determinants of transcriptional divergence in discrete tissues and cell types. However, how cis- and trans-effects act across continuous trajectories of cellular differentiation in vivo is poorly understood. Here, we quantify al ...
Berlin2024
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High-throughput transcriptomics is of increasing fundamental biological and clinical interest. The generation of molecular data from large collections of samples, such as biobanks and drug libraries, is boosting the development of new biomarkers and treatm ...
Objectives The endosymbiosis with Symbiodiniaceae is key to the ecological success of reef-building corals. However, climate change is threatening to destabilize this symbiosis on a global scale. Most studies looking into the response of corals to heat str ...