Figure 1. (A) Technologies enabling high throughput gene expression analysis. Breast cancer tumors are sampled at the treatment location and shipped to the central laboratory doing the assay, where pathologic review is done to assess cancer cell contents, followed by RNA preparation and integrity evaluation. Suitable samples are used to quantify RNA levels, thus assessing gene expression. When a gene is expressed, the transcription complex copies its DNA sequence into complementary RNA transcripts that are translated into proteins. High-throughput gene expression analysis aims at the quantification of messenger RNA (mRNA) populations in a given tissue. (B) Deoxyribonucleic acid microarray is the molecular biology technique enabling gene expression analysis in the MammaPrint assay. Ribonucleic acid is labeled with fluorescent dye and hybridized against thousands of different nucleotide sequences corresponding to different genes and arrayed on a solid surface (that is, a modified microscope glass slide). On hybridization, fluorescence emitted by single locations on the microarray is used to estimate gene expression levels. In MammaPrint a 2-color design is used, and RNA expression is estimated as a relative ratio between the sample and a reference RNA. For each patient, triplicate measurements are obtained from 2 microarray inverting the labeling scheme. (C) Real-time reverse transcriptase polymerase chain reaction is the enabling technology to assess gene expression in Oncotype DX and H/I tests. This technique is based on reverse transcription (see Glossary) of a specific mRNA into the complementary DNA molecule, which is used as a template in a polymerase chain reaction. The production of double-stranded DNA is accompanied by emission of light, which is recorded throughout the process and correlates to the amount of DNA that is produced. The input RNA is more concentrated in the initial reverse transcription reaction, and the earlier light is emitted during polymerase chain reaction, thus different levels of gene expression correspond to delayed light emission measurement (earlier vs. later) in the polymerase chain reaction step. (D) Gene-expression levels are translated by different mathematical transformation into indexes predicting disease recurrence (D).