Research reportThe use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia — caspase-3 as a case study
Introduction
A number of morphological and cellular changes take place in the affected area of the brain following experimental cerebral ischemia. These include the death of neurones and glia by programmed cell death, or apoptosis 4, 21, 23, 24, 25, 30. A characteristic feature of apoptosis is an increase in the RNA transcription for, and synthesis of specific proteins, including many of those involved in the cell death mechanism 10, 29.
The family of cysteine proteases known as caspases play a central role in apoptosis, contributing to a signalling cascade between death promoting stimuli and the cleavage of protein substrates which contribute to the characteristic apoptotic morphology 7, 28. During apoptosis inactive pro-enzyme forms of the caspases are cleaved and heterodimerise to form active enzymes. There is also increasing evidence of an upregulation of caspases at a transcriptional level during apoptosis in vitro 9, 26.
Several studies have now shown increased transcription of caspases, including caspase-3, in animal models of stroke 1, 3, 5, 14, 17, 27. These have employed a number of techniques including Northern blotting, semi-quantitative reverse transcription and polymerisation chain reaction (RT-PCR) and in situ hybridisation. These techniques do not allow a sensitive quantification of the relative levels of a particular transcript between samples. Recent advances in the use of fluorogenic probes in conjunction with PCR have enabled the measurement of an accumulating PCR product in real time 11, 13, 22. This allows the rapid generation of quantitative data showing changes in transcript numbers in tissue samples. An increase in throughput also allows a greater number of animals and experimental conditions to be included in a study, enabling more sensitive statistical analysis of the data.
The aim of the present study was to validate the use of the Taqman™ method of quantitative RT-PCR for measurement of changes in mRNA in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia in the rat and to use caspase-3 as a test case. This transcript has previously been measured in the same experimental model by Northern blotting, showing an increase in expression in the lesioned side of the brain [1]. We have carried out a detailed analysis of caspase-3 expression, comparing mRNA levels in the lesioned cortex to those observed in the contralateral cortex in ischemic animals and also the cortices of sham operated and naive rats. A thorough statistical analysis has been employed using data from individual rats. In the literature, a selection of housekeeping genes have been used to control for differences in RNA quantity and quality between samples. Here, we have examined the mRNA levels of a number of housekeeping genes and found differences in their expression patterns following the ischemic event. The data from these has been used to correct for discrepancies in RNA quality.
Section snippets
Materials and methods
Middle cerebral artery occlusion was carried out according to the method of Zea-Longa et al. [31]. Male Sprague–Dawley rats weighing 300–350 g were anesthetised with halothane. The left middle cerebral artery was occluded by an intraluminal filament at the origin from the Circle of Willis. The rats were euthanased 3, 6, 12 or 24 h after surgery by halothane overdose, following a neurological assessment to test the efficacy of the MCA occlusion. Parallel groups of rats either received sham
Results
The primary data output from the ABI prism 7700 sequence detector is the threshold cycle (CT), defined as the PCR cycle number at which the reporter dye fluorescence (ΔRn) is detectable above an arbitrary threshold. If this threshold is set within the range where ΔRn is increasing exponentially (Fig. 1a) then CT has a linear relationship with the logarithm of the copy number of the initial PCR template. An example of the data used to plot the standard curve for GAPDH is shown (Fig. 1b) together
Discussion
We have used a modified RT-PCR methodology to make precise measurements of comparative levels of transcripts of a series of genes in RNA samples derived from an animal disease model. This is possible because this technique allows the detection of an accumulating PCR product in real time. Comparisons between samples can thus be made during the exponential phase of the PCR, before reaction components become limiting. This technique also allows the use of standards; in this case, serial 10-fold
Acknowledgements
We would like to thank Dr. Andy Parsons for helpful discussions and critical reading of the manuscript.
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