Review
Control genes in quantitative molecular biological techniques: the variability of invariance

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Abstract

The measurement of transcript levels constitutes the foundation of today's molecular genetics. Independent of the techniques used, quantifications are generally normalised using invariant control genes to account for sample handling, loading and experimental variation. All of the widely used control genes are evaluated, dissecting different methodological approaches and issues regarding the experimental context (e.g. development and tissue type). Furthermore, the major sources of error are highlighted when applying these techniques. Finally, different approaches undertaken to assess the invariance of control genes are critically analysed to generate a procedure that will help to discern the best control for novel experiments.

Introduction

Control measurements form a fundamental tenet of all quantitative procedures. Determining the level of gene expression is no exception, whether this is performed by Northern-, southern-, slot- or dot-blotting, RNase protection-, nuclease protection- and in situ hybridization-assays, semi- and fully-quantitative PCRs all must employ appropriate controls as part of their experimental procedure. The primary control for these experiments is the parallel measurement of a control or housekeeping gene to assess differences in sample loading or reaction efficiencies, thus providing a means to evaluate and subsequently adjust for intrinsic experimental variations. This of course applies only to biological scenarios where transcription is active at all. In moribund or heavily histopathologically affected cells, for example, gene transcription may be reduced or indeed cease entirely. A similar situation exists in cells undergoing severe/acute stress (e.g. heat shock), where transcription is transiently blocked. In both of the examples mentioned, true control genes do not exist. In all other cases, pivotal control genes have to exhibit two major properties: firstly they should be essential for the maintenance of cellular function and viability (Finnegan et al., 1993), and therefore should be ubiquitously expressed in all tissues; and secondly their transcription should not be affected within the experimental context being investigated. For example, when comparing differential exposure regimes, developmental stages or tissue distributions, the expression of invariant control genes should remain at a steady-state level, whilst the transcription of target genes may be up- or down-regulated. Therefore, the use of internal controls facilitates the direct and sound measurement of comparative gene expression.

Identifying suitable control genes has been a major challenge in the field of molecular biology. Over the last decade, numerous candidate genes have been forthcoming, some of which have now established themselves as an integral part of quantitative approaches. However, to date no single gene has been awarded the unconditional approval by the scientific community. In many cases factors may modulate the expression of control genes, resulting in a slight variability of the so-called constitutively expressed housekeeping genes. Whilst these factors are often of unidentified origin, two prevailing reasons have been highlighted, namely the cross-reactive response to pseudo-genes and the presence of differentially expressed isoforms. This short review aims to bring together some key papers on the use and abuse of control genes assumed to be invariant.

Section snippets

The choice is yours!

To ensure that all samples contain equal amounts of starting material, it may seem reasonable to assess DNA/RNA concentrations by means of standard optical spectrophotometery. However, this physical technique has serious downfalls and thus is, by itself, not a feasible option. Values obtained are notoriously unreliable due to optical interference by varying amounts of contaminating material, such as ribosomal RNAs and transfer RNAs. This also the case during cDNA synthesis when excess reaction

Actin

Actin is one of the major components of cytoplasmic microfilaments in eukaryotic cells. It plays an important role in diverse cellular functions, such as cyctoplasmic streaming, changes in cell shape, cell motility, phagocytosis, cell division, the distribution of plasma membrane proteins and the generation of contractile force in both muscle and non-muscle cells (Romans et al., 1995; Kusakabe, 1997). As a member of a multigene family, every organism typically possesses three to four highly

Glyceraldehyde-3-phosphate dehydrogenase (G3PDH/GAPDH)

The glycolytic tetramer glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in cellular metabolism. Although it was initially believed that GAPDH is expressed as a single-copy nuclear gene (Bhatia, et al., 1994), genome sequencing data have identified at least two functionally independent GAPDH genes in Caenorhabditis elegans, Drosophila melanogaster and humans. Compared to actin, GAPDH's low copy number as well as the slightly less conserved intra-specific

Cyclophilin (CYP)

Cyclophilins, or peptidyl-prolyl cis-trans isomerases, are enzymes belonging to the superfamily of immunophilins. Their biological significance is manifested by the catalysis of protein folding via peptide bond rotation on the amino side of proline residues (Fischer et al., 1989; Takahashi et al., 1989), the action as a chaperone for protein trafficking as well as the nucleolytic degradation of the genome (Montague et al., 1997). Bio-medical interest was triggered by the discovery that CYP

Ribosomal subunits

The 18S and 28S ribosomal subunits are a further example of commonly used internal controls. A literature review of its uses revealed a reoccurring theme: when directly compared to other housekeeping genes they compare extremely favourably in terms of steady-state expression levels. Examples include gene expression studies conducted on normal, benign tumorigenic and highly metastatic human cell lines (Bhatia et al., 1994), rat liver tissues (Leeuw et al., 1989), analyses of transcription under

Other housekeeping genes

The elongation factor-1 alpha (EF-1 alpha) is an ubiquitous protein that binds aminoacyl-transfer RNA to ribosomes during protein synthesis. It has been stipulated to be a good invariant control to adjust for differences in tube-to-tube loading and/or degradation (Dostal et al., 1994). However, being an integral part of the translation apparatus, its expression can be modulated in areas of high protein turnover such as rapidly growing tissue, plant meristems and gametophytes (Ursin et al., 1991

By chance or design

Given the wide selection of possible control genes it is difficult to identify which is the most appropriate when designing a new experiment. The choice available will initially be determined by the experimental design, for although exploitation of ribosomal RNA may be a preferred option, use of Poly(T) separation chemistry or cDNA priming will eliminate this option. If ribosomal RNA is not an option then a review of all the appropriate literature concerning the experimental exposure regime and

Conclusions

It may well be scientifically acceptable to use any one of the major control genes and rarely will a referee question their applicability. However, the examples brought together in this short review clearly illustrate that no single gene has been shown to be invariant per se. Therefore, it is advisable to address the issue of the ‘variance of invariance’ particularly when dealing with novel biological material or when applying previously untested exposure regimes. The dangers of complacency are

Acknowledgements

We have tried to include the major recent findings, but have had to omit some reports due to space limitations and for this we beg indulgence from the authors. Furthermore we wish to acknowledge the continuous support from the British Natural Environmental Research Council (NERC) and the Royal Society.

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