The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia--caspase-3 as a case study.
Quantitative reverse transcription and polymerisation chain reaction (RT-PCR) using Taqman¿trade mark omitted¿ fluorogenic probes has been used to measure changes in gene expression in the cerebral cortex of rats in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of three housekeeping genes have been analysed in this model to determine which gene showed least change following experimental insult. In the lesioned cortex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin and 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% increase 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 sensitively and quantitatively than other available techniques and highlights the need for careful choice of control or housekeeping genes used for RNA comparisons.
Harrison
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DC
Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, UK. david_c_harrison@sbphrd.com
Medhurst
A D
AD
Bond
B C
BC
Campbell
C A
CA
Davis
R P
RP
Philpott
K L
KL
D000199:Actins / Q000235:genetics; D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000235:genetics*; D002540:Cerebral Cortex / Q000201:enzymology*; D007839:Functional Laterality; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D002546:Ischemic Attack, Transient / Q000201:enzymology*; D008297:Male; D020768:Middle Cerebral Artery; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D000199:Actins; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D019696:Peptidylprolyl Isomerase