Semiquantitative analysis of low levels of mRNA expression from small amounts of brain tissue by nonradioactive reverse transcriptase-polymerase chain reaction.
We have developed an easy and fast method to semiquantify low levels of mRNA from small amounts of brain tissues based on nonradioactive reverse transcription-polymerase chain reaction (RT-PCR). The regulation of mRNA for the growth associated protein GAP-43/B-50 and the homeodomain protein islet-1 was examined in the facial nucleus of the rat after a unilateral transection of the nerve. In both cases a similar sensitivity for radioactive and nonradioactive RT-PCR methods was found. The expression of the housekeeping gene, cyclophilin A was used to normalize total mRNA amounts and PCR conditions. After amplification the PCR products were separated electrophoretically on polyacrylamide gels. For nonradioactive semiquantification gels were stained with ethidium bromide and recorded using a CCD camera and transillumination. The recordings were evaluated with specialized software. Using nonradioactive RT-PCR, the increase in GAP-43/B-50 mRNA in response to axotomy was easily detectable in the small volume of tissue obtained from the facial nucleus. In contrast, the low expression of islet-1 mRNA made it necessary to develop a two-step amplification procedure in order to provide a reliable semiquantitative analysis. The procedure included preamplification of the cDNA and subsequent purification of the cDNA. Using this method, the down-regulation of islet-1 could be demonstrated with a similar sensitivity to that previously shown with radioactive RT-PCR.
Hager
G
G
Department of Neuromorphology, Max-Planck-Institute of Neurobiology, Martinsried, Germany. hager@neuro.mpg.de
Eckert
E
E
Schwaiger
F W
FW
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002138:Calibration; D005154:Facial Nerve / Q000378:metabolism*; D019922:GAP-43 Protein / Q000378:metabolism*; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015534:Trans-Activators / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D019922:GAP-43 Protein; C110528:Mapk8ip1 protein, rat; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D019696:Peptidylprolyl Isomerase