[Frontiers in Bioscience 14, 552-569, January 1, 2009]

Methods for quantitation of gene expression

Vigdis Nygaard, Eivind Hovig

Department of Tumor Biology, Institute for Cancer Research, Norwegian Radium Hospital, 0310 Oslo, Norway


1. Abstract
2. Measuring gene products at the transcriptional level
3. A reductionist approach versus a high throughput approach
4. Common methods for measuring mRNA levels
4.1. Subtractive hybridization
4.2. Differential display
4.3. RNase protection assay
4.4. Quantitative real time RT-PCR
4.5. Real competitive PCR
4.6. Serial analysis of gene expression (SAGE)
4.7. Massive parallel signature sequencing (MPSS)
4.8. Microarray technology
4.8.1. Principle of microarray technology and array platforms
4.8.2. Target preparation and hybridization
4.9. General technology comparison
5. Increasing technology applicability by resolving restrictions and limitations
5.1. Global mRNA amplification
6. Concluding remarks and future perspective
7. References


Gene expression of protein encoding genes can be quantitatively measured at the transcriptional level by a number of low- to high-throughput methods. The sensitivity of each method is dependent on both the intrinsic properties of the respective technology and the absolute number of each mRNA molecule to be measured. For these reasons, the correlation of measurements between technological platforms may be variable. Due to the complexity of the transcriptome, the purpose of a gene expression study dictates the choice of method as each is connected to a set of advantages and disadvantages. Strategies such as global mRNA amplification of small samples, have been implemented to overcome previous limitations. However, stochastic events will limit quantitative measurements of any tool when in-put levels are extremely low. Due to the versatile nature of microarray technology, this method will likely persist as a highly applied tool to query the levels of non-coding transcripts, a new expansion in the field of gene expression analysis although possible advances of the technology may occur.