User:Hogwild7/sandbox
Ribosome profiling is a technique that uses messenger RNA (mRNA) to determine what proteins are being translated. It produces a “global snapshot” of all the ribosomes active in a cell at a particular moment. Consequently, this enables researchers to identify the location of translation start sites, their distribution, and the speed of the translating ribosomes]]).[1]
History[edit]
Ribosome profiling was formed from the old discovery that the mRNA within a ribosome can be isolated through the use of nucleases that degrade unprotected mRNA regions. This technique analyzes the ratio of multiple specific mRNA’s to proteins being synthesized, to provide insight into global gene expression. Prior to its development, efforts to measure this ratio included microarray analysis on the RNA isolated from polysomes, as well as translational profiling through the affinity purification of epitope tagged ribosomes. However, neither of these techniques provides the positional and quantitative information of ribosome profiling.]]).[1]
Procedure[edit]
- Lyse the yeast cells and isolate the mRNA molecules bound to ribosomes.
- Immobilize complexes (commonly with cyclohexamine but other chemicals can be employed) and generate cross-linking using formaldehyde.
- Tag the mRNA-ribosome complexes in order to isolate them.
- Using nucleases, cut away all of the RNA not directly bound to the ribosome.
- Phenol/chloroform purification of mixture to remove proteins.
- Convert RNA to cDNA using reverse transcriptase.
- Sequence cDNA strand.
- Compare sequence results to chromosome strands to determine the regions that are most prevalently being translated.]]).[2]
Materials[edit]
- RNA-ribosome complexes
- Cyclohexamine
- Nucleases
- Phenol/Chloroform
- Reverse transcriptase
- dNTP
- Sequencing method-cDNA library.]]).[2]