Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching.
We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.
Lindqvist
Niclas
N
Department of Neuroscience, Biomedical Center, Uppsala University, Box 587, SE-751 23 Uppsala, Sweden.
Vidal-Sanz
Manuel
M
Hallböök
Finn
F
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D004210:Dissection / Q000295:instrumentation / Q000379:methods; D005260:Female; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000235:genetics*; D008860:Microscopy, Ultraviolet / Q000295:instrumentation / Q000379:methods; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D015203:Reproducibility of Results; D012160:Retina / Q000166:cytology / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013266:Stilbamidines; D013477:Superior Colliculi / Q000166:cytology / Q000378:metabolism; D014795:Visual Pathways / Q000166:cytology / Q000378:metabolism
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D013266:Stilbamidines; D020794:Receptor Protein-Tyrosine Kinases