Decreased beta-actin mRNA expression in hyperglycemic focal cerebral ischemia in the rat.
beta-Actin is often used as a housekeeping gene when performing reverse transcription-polymerase chain reaction (RT-PCR) analysis for cerebral ischemia models. In the present study, we tested two different control genes used for RT-PCR experiments, beta-actin and porphobilinogen deaminase (PBG-D), in a rat model of focal cerebral ischemia under normo- or hyperglycemic conditions. A three-vessel occlusion model with permanent middle cerebral artery occlusion was used in the rat. beta-Actin mRNA expression was decreased in hyperglycemic ischemic rats compared to normoglycemic ischemic animals 3 h post-ischemia. beta-Actin protein content was unchanged. As for PBG-D, its mRNA expression remained constant throughout the groups. Our data thus show that, following focal cerebral ischemia in hyperglycemic conditions, beta-actin is an unsuitable housekeeping gene whereas PBG-D is more appropriate. This study clearly demonstrates the importance of selecting a stable housekeeping gene when performing RT-PCR experiments.
Bémeur
Chantal
C
Laboratoire de Neurobiologie, Centre de Recherche du CHUM, Hôpital Notre-Dame, Montreal, QC, Canada H2L 4M1. chanchan@globetrotter.net
Ste-Marie
Line
L
Desjardins
Paul
P
Hazell
Alan S
AS
Vachon
Luc
L
Butterworth
Roger
R
Montgomery
Jane
J
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002545:Brain Ischemia / Q000150:complications / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005508:Food Deprivation; D005786:Gene Expression Regulation / Q000502:physiology*; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D006943:Hyperglycemia / Q000150:complications / Q000235:genetics / Q000378:metabolism*; D008297:Male; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
D000199:Actins; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011163:Hydroxymethylbilane Synthase