Every cell in an organism has identical genetic material (= genome). However, the different cells only ever use a subset of their genes, depending on the role the cell assumes in the organism. For example, muscle cells have mechanical properties, while cells in the pancreas produce insulin. In addition to these functional differences, the cell’s stage of development and even the time of day can influence the switching on and off of individual genes.
Individual cells of an organism can therefore differ greatly in their gene expression (=transcriptome), i.e. the transfer of genetic information into products (metabolics) that can be used by the cell or the organism. Gene expression is the synthesis of proteins from DNA. Gene expression analysis, also known as transcriptome analysis, measures which genes are switched on or off at a given time. Methods of gene expression analysis measure the concentration of the so-called messenger RNA (mRNA). mRNA is a single-stranded ribonucleic acid (RNA) that translates the genetic information in DNA into the structure of a specific protein in a cell.
For gene expression analysis, the cells or organisms of interest are first exposed to various experimental conditions (e.g. higher and lower temperatures) and the resultant changes in mRNA concentration are measured. A critical consideration in such experiments is having a sufficient number of technical and biological replicates, i.e. several identical repetitions per experimental condition, in order to rule out random effects as reliably as possible.
After the experiment, RNA is isolated from the organisms, tissues or cells. Since RNA is a very unstable molecule compared to DNA, and quickly degrades at room temperature, this step should ideally be carried out immediately after the experiment. Alternatively, the samples can be preserved in liquid nitrogen. After the RNA has been isolated, gene expression analysis is usually carried out using “RNA-Seq,” also known as whole-transcriptome shotgun sequencing. The nucleotide sequence of the RNA is determined using high-throughput methods (next-generation sequencing). For this purpose, the RNA is first enzymatically translated back into complementary DNA (cDNA) with a reverse transcriptase so that DNA sequencing, e.g. on an Illumina platform, can be carried out. The sequences obtained are then compared with a reference genome of the species (if available) or gene database in order to assign the sequences to the corresponding genes. The sets of genes from the replicates under the different experimental conditions can then be statistically compared to identify the genes that may be affected by the environmental condition being tested.