Ribosoomprofilering

Identifying regions in a genome that are transcribed and translated into proteins is a vital step in understanding a genome and its expression.

This is achieved with a technique called ribosome profiling, also referred to as ribo-seq. It maps the positions of ribosomes on mRNA and identifies mRNAs that are being actively translated into proteins.

To isolate the RNA, cells must first be lysed to access the molecules inside them. The lysate is then treated with RNases. These enzymes cleave the mRNAs that are not covered with ribosomes, leaving only the protected mRNA fragments.

The ribosome protected fragments are then separated from the unprotected, cleaved fragments using a sucrose gradient.

The ribosomes are then removed from the mRNA fragments, and the RNA are converted into DNA by RT-PCR, using the enzyme reverse transcriptase.

Next, the DNA is sequenced and mapped on the reference genome. This determines the exact location of the ribosome along each mRNA.

Ribosome profiling can also help to identify unrecognized open reading frames or ORFs. An ORF is a region of DNA between a start codon and a stop codon that can be translated into protein. Finding ORFs can be helpful in the id