Biochemistry Questions Biochemistry Questions / Describe the process of northern blotting and how it can be used to determine gene expression.

Northern blotting is a laboratory technique used to assay relative quantities of RNA in a sample, which can be used to determine relative gene expression.

A sample of RNA is separated by size through agarose gel electrophoresis. This separated RNA is then transferred to a membrane by blotting the gel, using a transfer buffer (which is normally negatively charged to have a high affinity for the RNA). Labelled probes are then used to image locations and relative quantities of RNA, typically with radioisotopes on X-ray or photographic paper.

A northern blot can be used to determine relative gene expression, based on the intensity of the bands present on the photographic paper. This method doesn’t allow for easy comparison between samples, although constitutively-expressed housekeeping genes can be used to normalise the band intensity and attempt to form comparisons between samples.

RNA-seq would be a more modern incarnation of this method, allowing cDNA sequencing to be used to accurately determine the transcriptional activity of genes. This gives more accurate data, due to the sequence of each transcript being determined, as well as giving precise values for the number of mRNA transcripts present in a sample.