METHODS


We used a differential display (DD) method termed READS to visualize global changes in gene expression. While DD is more time consuming we are more confidant of its results. In short DD uses a pool of the cell's CDNA's that have been cut with restriction enzymes then run through a gel to seperate the different genes. There is a whole slew of selection proceseses to maintain purity of the sample. The band on the gel representing the gene is analysed both qualitativly and quantitativly as well as being sequenced. The way we used this process we can only compare different expressions of the same gene under different circumstances. termed Differential Display (DD) to visualize global changes in gene expression. While DD is more time consuming we are more confidant of its results. In short DD uses a pool of the cell's CDNA's that have been cut with restriction enzymes then run through a gel to seperate the different genes. There is a whole slew of selection proceseses to maintain purity of the sample. The band on the gel representing the gene is analysed both qualitativly and quantitativly as well as being sequenced. The way we used this process we can only compare different expressions of the same gene under different circumstances.
This method has the advantage over other similar hybridization oriented methods is that the position of fragments corresponding to known genes is predictable and that no prior knowledge of the sequence is needed to detect previously "unknown" genes.

(click picture for expanded view)
Schematic of DD process


(click on picture to enlarge)
Typical DD gel
Most of the band intensities were quantified by eye,
confirmed by a second investigator, and
expressed as a single digit numeric. A part of the bands were quantified using
PhosphorImager System (Molecular Dynamics).

image obtained from www.genehunter.com
the following are some references: