Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media.
We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and beta-actin in cortical brain slices incubated for up to 24h in Ringer's solution and Dulbecco's modified Eagle's medium (DMEM) at 25 and 37 degrees C. Our data reveal an optimal temporal working window between 1 and 6h when slices are incubated in Ringer's solution at 25 degrees C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 degrees C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.
Dos-Anjos
Severiano
S
Area de Biología Celular, Department Biología Molecular, Instituto de Biomedicina, Universidad de León, 24071 León, Spain.
Martínez-Villayandre
Beatriz
B
Montori
Sheyla
S
Salas
Alberto
A
Pérez-García
Carlos César
CC
Fernández-López
Arsenio
A
D000818:Animals; D001921:Brain / Q000378:metabolism*; D003470:Culture Media; D004563:Electrochemistry; D005786:Gene Expression Regulation / Q000235:genetics*; D008297:Male; D008957:Models, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D046509:Tissue Culture Techniques / Q000379:methods*
D003470:Culture Media; D012333:RNA, Messenger