A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.
This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.
Shifman
M I
MI
Brain Research Laboratory, State University of New Jersey, Newark 07102, USA.
Stein
D G
DG
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D001923:Brain Chemistry / Q000502:physiology*; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism; D008163:Luminescent Measurements; D009414:Nerve Growth Factors / Q000096:biosynthesis*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats
D009414:Nerve Growth Factors; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D004076:Digoxigenin