Dc. Harrison et al., The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia - caspase-3 as a case study, MOL BRAIN R, 75(1), 2000, pp. 143-149
Quantitative reverse transcription and polymerisation chain reaction (RT-PC
R) using Taqman(TM) fluorogenic probes has been used to measure changes in
gene expression in the cerebral cortex of rats in the permanent middle cere
bral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of t
hree housekeeping genes have been analysed in this model to determine which
gene showed least change following experimental insult. In the lesioned co
rtex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin an
d glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not change. We have
also used this methodology to examine modulations in the level of caspase-3
mRNA during focal ischemia in the rat. Caspase-3 mRNA showed a 41% increas
e at 6 h post-MCAO, which was specific to the lesioned cortex. This change
became more pronounced with time, showing an increase of 220% at 24 h. This
methodology enables changes in mRNA expression to be analysed more sensiti
vely and quantitatively than other available techniques and highlights the
need for careful choice of control or housekeeping genes used for RNA compa
risons. (C) 2000 Elsevier Science B.V. All rights reserved.