Sensitive northern blot hybridization using digoxigenin RNA probes for the mRNA detection of two glucose transporter isoforms.
Diseases involving glucose metabolism disorders are more and more prevalent. Therefore the question of glucose transporter gene expression is being addressed in experimental and clinical studies. Radioactive probes are generally used to assess glucose transporter mRNA levels, but these probes are short-lived, costly and harmful to the environment. Alternative methods that do not present these disadvantages, for example digoxigenin (DIG) labelled probes, might prove to be very interesting for the study of glucose transporter mRNA. The aim of the present work was to compare DIG-labelled cRNA probes to 32P-labelled cRNA probes in order to see whether or not the non-radioactive method can be used to assess glucose transporter gene expression. This work shows that DIG-labelled glucose transporter (GLUT1 and GLUT4) cRNAs are suitable probes for the assessment of these gene expressions. We have found that the DIG system offers a much higher sensitivity than the 32P system for both GLUT1 and GLUT4 mRNA detection. This represents a decisive advantage in human studies where tissue quantity is a limiting factor. In addition, stability, safety, time saving and cost reduction are other considerations that make DIG-labelled GLUT1 and GLUT4 cRNAs very attractive.
Nivet
V
V
Biochemical Laboratory, Salpetri¿ere Hospital, Paris, France.
Hajduch
E
E
Hainault
I
I
Delattre
J
J
Lavau
M
M
Hainque
B
B
D000818:Animals; D015152:Blotting, Northern / Q000379:methods* / Q000706:statistics & numerical data; D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012680:Sensitivity and Specificity
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495679:Slc2a1 protein, rat; C495694:Slc2a4 protein, rat; D004076:Digoxigenin