A method utilizing differential culture and comparative RT-PCR for determining RNA expression in superior cervical ganglion neurones.
In order to assess the neuronal expression of caspase mRNA in primary cultures of rat superior cervical ganglion (SCG) neurones a method of differential cell purification and comparative RT-PCR was devised. SCG primary cultures generally contain variable percentages of non-neuronal contaminants, which influence RT-PCR results. We optimised a neuronal purification method, allowing the preparation of both highly purified neuronal cultures and mixed cultures, enriched in non-neuronal contaminants. These two sets of cells were cultured in parallel and subsequently analysed by RT-PCR. The use of cell type specific oligonucleotides allowed evaluation of the relative distribution of neuronal (neurofilament) and non-neuronal transcripts in the two cultures. In parallel, specific oligonucleotides were used to detect the mRNA levels of caspase family members. The partition of neurofilament transcript between pure and mixed cultures was found to be statistically different from the partition of the non-neuronal markers. Therefore statistical difference from the partition of non-neuronal markers was taken as evidence for expression in neurones. We show that caspase-2, -3, -6, -7 and -9 transcripts are expressed in SCG neurones whereas caspase-1 is probably absent. Furthermore, none of these transcripts are upregulated during neuronal death induced by nerve growth factor withdrawal. This method could be applied to the analysis of other transcripts in SCG and other primary neuronal cultures containing significant percentages of contaminant cell types.
Spadoni
Cesare
C
Eisai London Research Laboratories Ltd, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK.
Taylor
Joanne
J
Neame
Stephen
S
D000818:Animals; D015153:Blotting, Western; D020169:Caspases / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017783:Superior Cervical Ganglion / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*
D017931:DNA Primers; D012333:RNA, Messenger; D020169:Caspases