The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models.
TaqMan reverse transcription polymerase chain reaction (RT-PCR) is a recently developed technique which allows the measurement of an accumulating PCR product in real time. In the present study we have validated the use of TaqMan RT-PCR for mRNA localisation studies in human and rat tissues, and for the investigation of gene expression changes in CNS animal models. In human brain, D(2) receptor mRNA was enriched in caudate nucleus and putamen, whilst in rat brain, highest levels of D(2) receptor mRNA expression were observed in striatum and nucleus accumbens, consistent with the known distribution of this receptor in basal ganglia. In a rat model of permanent middle cerebral artery occlusion (pMCAO), endogenous interleukin-1 receptor antagonist (IL-1ra) mRNA was upregulated over 30-fold at 24 h post-lesion in both striatum and cortex ipsilateral to artery occlusion. Brain-derived neurotrophic factor (BDNF) mRNA was transiently upregulated 3.7-fold at 3 h, but not at 24 h or 3 days after induction of cortical spreading depression (CSD) in rats. Our observations in these two animal models using TaqMan RT-PCR were consistent with previous reports using other techniques. In conclusion, TaqMan RT-PCR assays provide a rapid and reliable method for semi-quantitative analysis of gene expression in the nervous system.
Medhurst
A D
AD
Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Third Avenue, Essex, CM19 5AW, Harlow, UK. andy_medhurst-1@sbphrd.com
Harrison
D C
DC
Read
S J
SJ
Campbell
C A
CA
Robbins
M J
MJ
Pangalos
M N
MN
D000199:Actins / Q000235:genetics; D000818:Animals; D001923:Brain Chemistry / Q000235:genetics*; D013181:Cortical Spreading Depression; D017931:DNA Primers; D004195:Disease Models, Animal; D015870:Gene Expression / Q000502:physiology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics* / Q000503:physiopathology; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D000199:Actins; D017931:DNA Primers; D012333:RNA, Messenger; D017448:Receptors, Dopamine D2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019914:Taq Polymerase; D019696:Peptidylprolyl Isomerase