2001, pp 281-291

Development and Validation of an Externally Standardised Quantitative Insulin-like Growth Factor-1 RT-PCR Using LightCycler SYBR Green I Technology

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Abstract

The cytokine insulin-like growth factor-1 (IGF-1) is considered to mediate anabolic growth hormone actions in various tissues and species. During postnatal growth, IGF-1 stimulates protein synthesis and improves glucose utilisation [7, 3]. In addition, locally expressed IGF-1 is an important growth regulator acting in an auto- and paracrine manner [12]. To investigate local tissue-specific expression even in tissues with low abundancies, a very sensitive method is required which allows for reliable quantification of IGF-1 mRNA. Because of its high sensitivity, reverse-transcription with subsequent polymerase chain reaction (RT-PCR) is being increasingly used to quantify physiologically relevant changes in gene expression. RT-PCR has a detection limit 10–100 fold lower than other methods, e.g. protection-assay or northern-hybridisation, respectively [11]. The RT-ribonuclease PCR quantification technique of choice depends on the target sequence, the expected range of the mRNA amount present in the tissue, the degree of accuracy required, and whether quantification needs to be relative or absolute. Externally standardised RT-PCR with quantification on ethidium bromide stained gels followed by densitometry is widely used, but the degree of accuracy is limited and the quantification is more relative than absolute [10]. For an exact quantitative measurement of low abundant gene expression only a few PCR methods allow reliable mRNA quantification. At present the following RT-PCR methods are suitable for sensitive quantification:

  1. Internally standardised competitive RT-PCR measured by HPLC separation and UV detection [9] or high resolution gel electrophoresis followed by densitometric analysis [8]: In a competitive RT-PCR, a reference RNA mutant is reverse transcribed and co-amplified in the same reaction tube with the native mRNA sequence of interest. Internally standardised RT-PCR is a very time-consuming and laborious technique. It is generally believed to yield the most precise results, because all parameters throughout RT-PCR act on both the analyte and reference mutant.

  2. Externally standardised RT-PCR with online-detection using LightCycler SYBR Green I technology [6, 13]: LightCycler PCR with SYBR Green I online detection produces reliable and rapid results. Because it uses an external standard curve, the amplification efficiencies for the calibration curve and the analyte must be equal for accurate quantification.

  3. Externally standardised RT-PCR with online-detection using specific LightCycler hybridisation probes [14]: This detection format is based on fluorescence resonance energy transfer.