Characterization of mRNA expression in single neurons.
How neurons differ from each other is largely determined by their specific repertoire of mRNAs. The genes expressed by a given neuron reflect its developmental history, its interaction with other cells, and its synaptic activity. Since the introduction of reverse transcription polymerase chain reaction (RT-PCR), it has been possible to identify specific mRNAs present in small samples of total RNA. But isolating RNA from only those cells of interest, and not others, represents a significant challenge. Several approaches can be used to isolate RNA from selected neurons. Following whole-cell patch-clamp recording, mRNA can be harvested from living cells by aspirating the cytoplasm into the patch-clamp pipette. Transcripts expressed in the recorded neuron can then be amplified by RT-PCR. Another way of isolating identified neurons is to use cell-specific promoters to drive the expression of a marker gene such as green fluorescent protein (GFP). RNA can then be isolated from GFP-positive cells. In a tissue context, laser microdissection can also be used to excise the cells of interest directly into an RNA isolation solution. The above methods of RNA isolation can also be combined with RNA amplification and microarray technology to identify specific transcripts that are unique to the cell type being studied. Here we provide detailed protocols for harvesting RNA from single cells, methods for RNA purification, and PCR amplification.
Lin
David M
DM
Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.
Loveall
Brandon
B
Ewer
John
J
Deitcher
David L
DL
Sucher
Nikolaus J
NJ
D000818:Animals; D002469:Cell Separation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D018408:Patch-Clamp Techniques / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D005819:Genetic Markers; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins