Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons.
Identification of neurons for single-cell mRNA profiling is difficult when cells of interest are located in heterogeneous brain regions. We developed a protocol in which acutely dissociated neurons are immunocytochemically labeled prior to single-cell reverse transcription-polymerase chain reaction (RT-PCR). We tested the protocol on hypothalamic melanin-concentrating hormone (MCH) and prepro-orexin (PPO) neurons, which are similarly distributed but functionally different. Cells dissociated from the perifornical region of the posterior hypothalamus of juvenile or adult rats were incubated with anti-MCH or anti-PPO primary antibodies, followed by washout and incubation with fluorescein-tagged secondary antibodies. Individual labeled cells were subjected to RT-PCR with primers for PPO and MCH. MCH mRNA was detected in 26 out of the 38 successfully reverse-transcribed cells identified as MCH-containing, and 28 cells out of the 42 identified as PPO-containing expressed PPO mRNA. No cell expressed both mRNAs. Most MCH neurons tested (five out of six) expressed the adrenergic alpha2A receptor mRNA, whereas it was absent from all seven PPO neurons tested. Neither PPO (n = 11) nor MCH (n = 6) cells expressed the type 2 orexin receptor mRNA. Thus, the method allows, with at least 66% confidence, immunocytochemical cell identification prior to mRNA studies of single neurons located in heterogeneous brain regions.
Volgin
Denys V
DV
Department of Animal Biology 209E/VET, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6046, USA. dvolgin@vt.upenn.edu
Swan
Jennifer
J
Kubin
Leszek
L
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D007028:Hypothalamic Hormones / Q000235:genetics / Q000378:metabolism; D007034:Hypothalamus, Posterior / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D008543:Melanins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D064446:Orexin Receptors; D000068797:Orexins; D010907:Pituitary Hormones / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D007028:Hypothalamic Hormones; D047908:Intracellular Signaling Peptides and Proteins; D008543:Melanins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010907:Pituitary Hormones; D011498:Protein Precursors; D012333:RNA, Messenger; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C019463:melanin-concentrating hormone