Competitive quantitative measurement of the AMPA receptor gene expression at the single cell level.
Our laboratory has developed a competitive reverse transcriptase polymerase chain reaction (RT-PCR) procedure to analyse the mRNA expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in single cells. By the use of an internal RNA standard competing equally with the four subunit's mRNA, we have analysed 283 whole single hippocampus CA1 cells from adult rat brain. The cells were sampled from three groups of animals: one control group, one group subjected to preconditioning ischemia, and one group subjected to global cerebral ischemia. After reverse-transcription and PCR-amplification of mRNA in the cells, the PCR product was digested using subunit specific endonucleases and quantified by Cy-5 fluorescence. The median mRNA copy numbers achieved from control rats were 290, 247, 207, and 16 GluR1-4, respectively.
Alsbo
Carsten W
CW
Laboratory of Molecular Neuropathology, Institute of Molecular Pathology, University of Copenhagen, 11 Frederik V's vej, DK-2100 Copenhagen, Denmark.
Wrang
Maria L
ML
Henrik Diemer
Nils
N
D000818:Animals; D002545:Brain Ischemia / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D002232:Carbocyanines; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004720:Endonucleases; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000502:physiology*; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D019194:Ischemic Preconditioning; D008297:Male; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D012319:RNA Polymerase II / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002232:Carbocyanines; D004720:Endonucleases; D005456:Fluorescent Dyes; D012319:RNA Polymerase II; D012333:RNA, Messenger; D018091:Receptors, AMPA; C085321:cyanine dye 5; C104723:glutamate receptor ionotropic, AMPA 3; C104724:glutamate receptor ionotropic, AMPA 4; C104722:glutamate receptor ionotropic, AMPA 2