A versatile Immuno-LCM/RNA-Seq method uncovers the rodent gonadotropin-releasing hormone neuron transcriptome
We developed and validated an Immuno-LCM/RNA-Seq method for transcriptome profiling of immunohistochemically labeled neurons. Immuno-LCM/RNA-Seq allows cell type-specific and high-throughput spatial transcriptomic studies without species limitations to genetically-modified mice. Innovative RNA-preserving strategies we introduced efficiently maintain RNA integrity in floated sections of formaldehyde-fixed rodent brains during both long-term tissue storage and immunohistochemical processing. Gonadotropin-releasing hormone (GnRH) neurons regulating reproduction were immunostained and harvested with laser-capture microdissection (LCM) for RNA isolation, random primer-based cDNA library preparation and bulk sequencing. The 13,000-14,000 transcripts we detected in GnRH neurons of rats and mice included 28 mRNAs implicated previously in human infertility. Interestingly, GnRH neurons exclusively expressed estrogen receptor alpha in mice and estrogen receptor beta in rats, among other unexpected species differences. Immuno-LCM/RNA-Seq is a versatile, sensitive and accurate bulk sequencing approach to characterize the transcriptome landscape of GnRH neurons and other neuronal phenotypes identified with immunohistochemistry in formaldehyde-fixed tissue sections of any species.