1. ¹Centre for Infectious Diseases and International Health, University College London, London, UK
2. ²Royal Free Medical School, London, UK
3. ³Department of Thoracic and HIV Medicine, Royal Free Hospital, London, UK
4. ⁴Centre for Academic Surgery, Barts and the London, Queen Mary's School of Medicine and Dentistry, London, UK

Correspondence: Dr J Huggett, Centre for Infectious Diseases and International Health, University College London, Windeyer Institute of Medical Research, 46 Cleveland St, London W1T 4JF, UK. E-mail: j.huggett@ucl.ac.uk

Received 1 November 2004; Revised 20 December 2004; Accepted 21 December 2004; Published online 7 April 2005.

Abstract

Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.